Synthesis and Characterization of 1H-Imidazole-4,5-dicarboxylic Acid-Functionalized Silver Nanoparticles: Dual Colorimetric Sensors of Zn2+ and Homocysteine.

A colorimetric assay has been developed for Zn2+ and homocysteine (Hcy) detection using functionalized silver nanoparticles (AgNPs). AgNPs have been synthesized using silver nitrate, where sodium citrate is used as a stabilizing agent and NaBH4 as a reducing agent. Then, the nanoparticles (citrate@AgNPs) were functionalized with 1H-imidazole-4,5-dicarboxylic acid (IDCA). UV-visible and FTIR spectra suggested that IDCA was functionalized on the surface of citrate@AgNPs through the N atom of the imidazole ring. The IDCA-functionalized silver nanoparticles (IDCA@AgNPs) simultaneously detected Zn2+ and Hcy from aqueous solution and showed different responses to the two analytes (Zn2+ and Hcy) based on the aggregation-induced color change of IDCA@AgNPs. They showed the color change from yellow to red, which was easily discriminated by visual inspection as well as UV-visible spectroscopy. The surface plasmon resonance absorbance values of Zn2+ and Hcy are 485 and 512 nm, respectively, when Zn2+ and Hcy react with IDCA@AgNPs. IDCA@AgNPs showed linearity with Zn2+ and Hcy concentrations, with the detection limit of 2.38 μM and 0.54 nM, respectively (S/N = 3). The IDCA@AgNPs showed excellent selectivity toward Zn2+ and Hcy compared to the different metal ions and amino acids, respectively. Optimal detection was achieved toward Zn2+ and Hcy in the pH range 3-10. In addition, IDCA@AgNPs were used to detect Zn2+ and Hcy from lake water, showing low interference.

Introduction

Zinc is the second most abundant transition element after iron, which participates in various aspects in biological metabolism. It plays various roles in the synthetic routes within cells,[1] normal growth of human body,[2] and normal brain function.[3] It is normally nontoxic, but uptake by humans due to high environmental concentrations of Zn2+ ion causes pulmonary manifestations, fever, chills, and gastroenteritis. Thus, the determination of trace amounts of Zn2+ ion is currently of great interest in medical sciences as well as environmental monitoring.

On the other hand, Hcy is a sulfur-containing amino acid in which a free −SH group is present as in cysteine, and Hcy is the intermediate formed during the conversion of methionine to cysteine. Free Hcy is not available naturally as it exists in plasma, in disulfide form as well as in protein-bound form. Therefore, it is complicated to determine the total Hcy after the reduction of disulfide. The normal concentration of Hcy in plasma ranges from 5 to 15 μM. The condition when the level of Hcy is above 15 μM is defined as hyperhomocysteinemia. High plasma levels of Hcy are associated with several diseases such as cardiovascular disease,[4] Alzheimer’s disease,[5] neural tube defects,[6] and osteoporosis.[7] Hence, the determination of Hcy in plasma or protein is highly important.

Currently, there are common techniques employed for the detection of Hcy or Zn2+, such as atomic absorption spectroscopy (AAS),[8] fluorescence,[9] electrochemical methods,[10] inductively coupled plasma mass spectrometry (ICP–MS),[11,12] and isotopic dilution mass spectrometry,[13,14] which provide sensitive, selective, and reproducible results, but they typically require expensive and sophisticated instruments, highly expensive that they are unsuitable for on-site analyses.

To contrast the abovementioned laboratory techniques, nanoparticle-based colorimetric sensors provide low-cost, simple monitoring and visual observation.[15] Among the various metallic nanoparticles, AgNPs are one of the most important other than gold nanoparticles due to the greater molar extinction coefficient (100-fold greater compared to gold), which improves the visibility and sensitivity when using absorption spectroscopy. Ihsan et al.[16] reported the synthesis of biologically synthesized colorimetric assay for selective determination of Zn2+ but not clearly study the selectivity test. Level-free silver nanoparticles were synthesized by K.-B. Lee and his co-worker[17] who reported the selective detection of Zn2+ but not in the presence of different pH of the medium. Karthiga and Anthony[18] reported the green synthesis of silver nanoparticles using leaf extracts for the determination of metal cations (Hg2+, Pb2+, Zn2+, and Co3+) over a wide range of pH. Furthermore, some reports were found using functionalized gold nanoparticles[19,20] for the colorimetric detection of Zn2+. To the best of our knowledge, very few reports were found for the determination of Zn2+ ion using metallic nanoparticles, especially AgNPs and AuNPs. Therefore, there is enough scope to develop colorimetric sensors using AgNPs. On the other hand, a simple, portable detection method is really a challenge for the determination of Hcy from aqueous solution. Leesutthiphonchai et al.[21] reported the selective determination of Hcy levels in human plasma using a silver nanoparticle-based colorimetric assay. Uehara reported[22] the colorimetric assay of Hcy using gold nanoparticles conjugated with thermoresponsive copolymers. Thus, there are very few reports on the colorimetric sensing of Hcy using functionalized metallic nanoparticles.

Recently, almost all surface modifications of AgNPs are based on Ag–thiol interactions.[23,24] Because of the strong binding affinity between Ag and the sulfur atom of the ligands to produce Ag–S covalent bonds,[25] undesirable aggregations are expected during the modification and centrifugation separation steps. The stability of AgNPs is really a challenge for the long-term. In the present study, we report IDCA-functionalized AgNPs from the as-prepared citrate-capped AgNPs; as the N donors of IDCA have stronger affinity with AgNPs compared to the carboxyl group[26] without involving any complicated surface modification and tedious separation/purification processes, we envision that the IDCA-functionalized AgNPs might be suitable for the development of a highly selective and sensitive assay for the colorimetric dual sensing of Zn2+ and Hcy from aqueous solution.

Synthesis of Functionalized Silver Nanoparticles

Silver nanoparticles (AgNPs) were prepared using NaBH4 as a reducing agent and sodium citrate as a stabilizer according to a previously reported method with slight modifications.[27,28] Sodium citrate solution (10 mL, 50 mM) was added into a conical flask (500 mL), which contained silver nitrate solution (250 mL, 0.25 mM), under vigorous stirring for 40 min. Then, NaBH4 (10 mL, 25 mM) was slowly added to the above solution at room temperature and stirred for 1 h. The dark colloidal solution changed to yellow, signifying the formation of citrate-capped AgNPs (Scheme a). To modify the surface of citrate-capped AgNPs, 100 mL of the as-prepared AgNP solution was taken in a 250 mL conical flask. Then, 0.078 g (0.5 mM) of 1H-imidazole-4,5-dicarboxylic acid (IDCA) (0.50 mM) was taken in 1 mM NaOH solution (10 mL, pH = 7.54) and added to the as-prepared 100 mL citrate@AgNP solution. Then, the mixture was stirred for 24 h at 60 °C, and the solution was cooled to room temperature. The IDCA@AgNPs (Scheme b) were obtained through centrifugation (25 min, 15,000 rpm) after removing the excess NaOH, IDCA, and citrate.

Scheme 1

Synthesis of (a) Citrate@AgNPs and (b) IDCA@AgNPs after Centrifugation for 24 h and 60 °C

Results and Discussion

Characterizations

Silver nanoparticles were prepared with citrate as the stabilizer and sodium borohydride as the reducing agent for AgNO3. IDCA was added to the AgNP solution for surface functionalization. The UV–visible absorption spectra of citrate@AgNPs and IDCA@AgNPs are shown in Figure a,b. In Figure b,a slight red shift is observed from 393 to 397 nm, indicating that IDCA was modified on the surface of AuNPs as compared to that of the unmodified citrate@AgNPs. Moreover, we carried out control experiments using the citrate@AgNPs, which did not show any response to 50 μM Zn2+ and 1 μM of Hcy (Figure S1, Supporting Information). These results proved that the IDCA@AgNPs have sensing function in contrast to the citrate@AgNPs. Upon the addition of 50 μM Zn2+ or 1 μM Hcy to IDCA@AgNP solution, its color readily changes from yellow to red, due to the binding of Zn2+ or Hcy with IDCA, decreasing the distance of AgNPs, yielding both a substantial shift in the plasmon band energy to a longer wavelength and a yellow-to-red color change (Figure c,d).

Figure 1

UV–vis spectra and photographic images (insert) of (a) citrate@AgNPs, (b) AgNPs@IDCA, (c) AgNPs@IDCA-Zn2+, and (d) AgNPs@IDCA-Hcy.

To understand the nature of binding mode by IDCA on AgNP surfaces, we studied the FTIR spectra. The FTIR spectra of citrate@AgNPs and IDCA@AgNPs are shown in Figure . It is evident from Figure a that the characteristic peaks of citrate@AgNPs are at 1600 cm–1 (υas, C=O) and 1329 cm–1 (υs, C–O), and a broad peak is around 3200–3420 cm–1 (O–H). The FTIR spectra of IDCA@AgNPs (Figure b) are similar to those of citrate@AgNPs. However, the difference is due to the appearance of two new peaks at 1400 cm–1 (C=N) and 3445 cm–1 (N–H stretching). These two new peaks are the characteristic peaks of the imidazole group in IDCA@AgNPs. Another new peak is present at 687 cm–1, which is due to the Ag–N bond,[29] and this peak is absent in citrate@AgNPs (Figure a). Thus, FTIR and UV–vis spectra clearly confirmed that the IDCA ligand functionalized with the citrate@AgNP surface through the N atom of the imidazole ring of IDCA.

Figure 2

FTIR spectra of (a) citrate@AgNPs and (b) IDCA@AgNPs.

In order to confirm the morphology and size, we performed the dynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses of citrate@AgNPs. The particle sizes of citrate@AgNPs ranged from 6 to 25 nm, and most of the particle sizes fell in the range of 8–15 nm, as evident from DLS (Figure a′), which is similar to the size distribution observed in the TEM images (Figure a). The DLS study and TEM are also performed for IDCA@AgNPs after the modification of citrate@AgNP surfaces. The particle sizes of IDCA@AgNPs ranged from 6 to 28 nm, and most of the particle sizes fell in the range of 9–21 nm, as evident from DLS (Figure b′), which is similar to the size distribution observed in TEM images (Figure b). The HRTEM images display the evidence of Zn2+- and Hcy-induced aggregation of IDCA@AgNPs, as shown in Figure c,d, and their corresponding size distribution was also characterized by DLS (Figure c′,d′). With the addition of Zn2+, the particle sizes ranged from 15 to 125 nm, and most of the particle sizes fell in the range of 25–80 nm, which is similar to the size distribution trends observed in the TEM images (Figure c). This observation indicated that Zn2+ induced the aggregation of IDCA@AgNPs. Similarly, with the addition of Hcy, the particle sizes ranged from 30 to 150 nm, and most of the particle sizes fell in the range of 40–90 nm. Thus, IDCA@AgNPs aggregate after binding with Hcy. The color change of AgNPs is highly sensitive to the size, shape, capping agents, medium refractive index, as well as the aggregation state of AgNPs.[30] Thus, change of color means variation of the size of AgNPs. When ligands as well as metal ions bind with AgNPs, “interparticle cross-linking mechanism”[31] occurs, resulting in the variation of color as well as size. When IDCA@AgNPs bind with Zn2+ and Hcy, resulting in the aggregation of AgNPs, various size ranges of particles were observed.

Figure 3

TEM images and their corresponding DLS of citrate@AgNPs (a,a′), IDCA@AgNPs (b,b′), IDCA@AgNPs-Zn2+ (c,c′), and IDCA@AgNPs-Hcy (d,d′).

Influence of pH on IDCA@AgNPs

To investigate the pH range in which the IDCA@AgNPs can effectively detect Zn2+ or Hcy, a pH titration was carried out. The effect of pH on the absorption band of IDCA@AgNPs is shown in Figure S2 (Supporting Information), showing the stability of the prepared IDCA@AgNPs with respect to different pH values of the solution. From Figure S2, it is evident that the prepared IDCA@AgNPs are stable in the pH range from 3 to 10, but they are unstable and aggregated when pH > 10 or pH < 3. This is due to the chemical changes taking place in the IDCA@AgNP solution in the surroundings of particles; at pH < 3, all carboxylates are protonated, so the particles aggregate, whereas at pH > 10, the carboxyl groups of IDCA remain deprotonated and the hydroxyl groups in the solution can overwhelm the surface of AgNPs, which also reflect in the corresponding color of IDCA@AgNPs (Figure S2, Top). Therefore, for the monitoring of Zn2+ ion or Hcy, the pH range 3–10 is suitable and used. The pH of the mother solution (IDCA@AgNPs) was adjusted using phosphate-buffered saline (PBS), and the pH was around 5.4. We studied the pH for the determination of Zn2+ ion, and it is shown in Figure a. The suitable pH for the detection of Zn2+ ion is the range of 4–10, and the color of the IDCA@AgNP solution also reflects the result of detection (Figure a, left top). Addition of Zn2+ in IDCA@AgNPs resulted in a high aggregation at pH 4–10; simultaneously, a new band appeared. At pH > 10, immediate decolorization of IDCA@AgNPs occurred, and the band disappeared because of the formation of colloidal Zn(OH)2.[32] At pH < 3, similar change was observed because in more acidic conditions the competition between H+ and Zn2+ ions with the free carboxylic groups of IDCA@AgNPs rendered the aggregation of IDCA@AgNPs. Therefore, the optimal pH range for detecting Zn2+ by IDCA@AgNPs is 3–10 (Figure ). Almost similar trends followed when Hcy was added (1 μM) onto IDCA@AgNPs at different pH (Figure ). However, the suitable pH range for the detection of Hcy is 4–9, and the color of the IDCA@AgNP solution also reflects the result of detection (Figure , top, right).

Figure 4

Photographic images (left, top) and absorbance spectra (left, bottom) of IDCA@AgNPs in the presence of Zn2+ ion in the pH range 1–12 and photographic images (right, top) and absorbance spectra (right, bottom) of IDCA@AgNPs in the presence of Hcy in the pH range 1–12.

Sensitivity of IDCA@AgNPs toward Zn2+ and Hcy

The sensing ability of IDCA@AgNPs with metal ions and various amino acids in aqueous solutions was tested. To evaluate the selectivity of IDCA@AgNPs toward various metal ions and various amino acids, the UV–vis absorption spectra of IDCA@AgNPs were observed in the presence of several metal ions (Zn2+, Cr3+, Hg2+, Cd2+, Pb2+, Cu2+, Ni2+, Co2+, Fe2+, Fe3+, Ca2+, Mg2+, K+, and Na+) and various amino acids (Hcy, Cys, Glu, Asp, Gly, His, Try, Lys, Ala, Cret, Theo, Phy-ala, Ser, and Met). Figure shows the effect of metal ions on the appearance of IDCA@AgNPs in solution. Zn2+ was the only ion that resulted in an absorption peak shift from 397 to 482 nm. This red shift could also be observed by the naked eye as a color change from yellow to red. Other metal ions did not influence the absorption spectra, indicating that no aggregation occurred. Thus, IDCA@AgNPs showed excellent sensitivity to Zn2+ ion over different metal ions. On the other hand, we tested the sensing ability of IDCA@AgNPs toward various amino acids such as Hcy, Cys, Glu, Asp, Gly, His, Try, Lys, Ala, Cret, Theo, Phy-ala, Ser, and Met. Figure shows the effect of amino acids on the appearance of IDCA@AgNPs in solution. Hcy was the only ion that resulted in an absorption peak shift from 397 to 512 nm. This red shift could also be observed by the naked eye as a color change from yellow to red. Other amino acids did not influence the absorption spectra, indicating that no aggregation occurred. Thus, IDCA@AgNPs detect Zn2+ and Hcy from aqueous solution.

Figure 5

Photographic images (top) and UV–vis spectra (bottom) of IDCA@AgNPs in the presence of different metal ions (50 μM).

Figure 6

Photographic images (top) and UV–vis spectra (bottom) of AgNPs in the presence of different amino acids (4 μM).

For addressing the sensitivity of the colorimetric assay, we choose two different wavelengths (for Zn2+: 397 and 485 nm; and for Hcy: 397 and 512 nm). The inset of Figure shows a linear relationship (R2 = 0.97743) between the absorption ratios (A485/A397) of IDCA@AgNPs with the concentration of Zn2+ over the range from 1 to 500 μM. From Figure , it is evident that the degree of aggregation of IDCA@AgNPs depends on the concentration of Zn2+ ions. The absorption spectra changed with the addition of different concentrations of Zn2+, and the change of color of IDCA@AgNPs could also be determined by naked eye when the concentration of Zn2+ ion was 50 μM (Figure S3, Supporting Information). The limit of detection for Zn2+ was found to be 2.38 μM (Figure S4, Supporting Information). On the other hand, we developed analytical data for the quantification of Hcy from aqueous solution. As shown in Figure , the UV–visible spectra of IDCA@AgNPs show a strong SPR peak at 397 nm; however, it is gradually decreased with the generation of a new SPR peak at 512 nm as the concentration of Hcy increased from 0.1 to 25 μM. Furthermore, the development of the color change from yellow to red depended on the increasing concentrations of Hcy, and the concentration of Hcy at 1 μM could be rapidly determined with the naked eye (Figure S5, Supporting Information). The insert of Figure shows a linear relationship (R2 = 0.96699) between the absorption ratio (A512/A397) of IDCA@AgNPs and the concentration of Hcy over the range from 0.1 to 25 μM. The limit of detection of Hcy was found to be 0.54 nM (Figure S6, Supporting Information). These results demonstrated that a higher concentration of Hcy induced a higher degree of IDCA@AgNP aggregation. Our work on IDCA@AgNPs is compared with other relevant works (Table S1, Supporting Information), and it is found that our probe (IDCA@AgNPs) is more efficient with respect to other probes.

Figure 7

Change of absorption spectra of IDCA@AgNPs in the presence of various concentrations of Zn2+ ion (1.0–500 μM).

Figure 8

Change of absorption spectra of IDCA@AgNPs in the presence of various concentrations of Hcy (0.1–25 μM).

In order to study the influence of other metal ions on Zn2+ binding to IDCA@AgNPs, competitive experiments were carried out in the presence of Zn2+ (200 μM) with other metal ions such as Hg2+, Cd2+, Pb2+, Cu2+, Ni2+, Co2+, Fe2+, Fe3+, Cr3+, Ca2+, Mg2+, K+, and Na+ at 500 μM (Figure ). The UV–vis spectra of the above interference ions are shown in Figure S7 (Supporting Information). The SPR absorption shift caused by the mixture of Zn2+ with another metal ion was similar to that caused solely by Zn2+. This indicates that other metal ions did not interfere in the binding of IDCA@AgNPs with Zn2+.

Figure 9

Absorbance ratios (A485nm/A397nm) upon the addition of IDCA@AgNPs to Zn2+ for the selected metal ions. Red bars represent the addition of a single metal ion (200 μM), and gray bars are the addition of Zn2+ (200 μM) with another metal ion (500 μM).

On the other hand, we studied the influence of various amino acids on Hcy (4 μM) binding by IDCA@AgNPs; selective experiments were carried out in the presence of several other amino acids such as Cys, Glu, Asp, Gly, His, Try, Lys, Ala, Cret, Theo, Phy-ala, Ser, and Met at 25 μM (Figure ), and their corresponding UV–vis spectra are shown in Figure S8 (Supporting Information). The SPR absorption shifts caused by the mixture of Hcy with another amino acid were similar to that caused solely by Hcy. This indicates that other metal ions did not interfere in the binding of IDCA@AgNPs with Hcy.

Figure 10

Absorbance ratios (A512nm/A397nm) upon the addition of IDCA@AgNPs to Hcy for the selected amino acids. Red bars represent the addition of a single amino acid (4 μM), and gray bars are the addition of Hcy (4 μM) with other amino acids (25 μM).

Sensing Mechanism for Binding Zn2+ and Hcy

To evaluate the sensing mechanism by IDCA@AgNPs toward Zn2+ and Hcy, a proposed schematic illustration is shown in Scheme . It is evident (Scheme ) that in IDCA@AgNPs there are two types of binding groups which are imidazole nitrogen and carboxylic group of IDCA. However, it is clearly noticed (Figures and 2) that the nitrogen atom of the imidazole group is utilized for the surface modification of citrate@AgNPs. Thus, in the presence of the deprotonated acidic group (COO–) of IDCA, both Zn2+ and Hcy can bind simultaneously, resulting in the different aggregation of IDCA@AgNPs in solution, changing the color from yellow to red. To clarify the involvement of the COO– group for simultaneously binding with Zn2+ and Hcy, FTIR spectra were recorded by mixing Zn2+ (200) and Hcy (2 μM) onto IDCA@AgNPs and are shown in Figure S9 (Supporting Information). Figure S9 clearly indicates that the original FTIR peak position of the carboxylic group in IDCA@AgNPs (Figure ) was 1601 cm–1 in the absence of Zn2+ as well as Hcy. However, in the presence of Zn2+ and Hcy, the peak position shifted to 1578 and 1552 cm–1 for Zn2+ and Hcy, respectively. The binding of Hcy may be due to the participation of carboxylate oxygen of IDCA through the weak hydrogen-bonding interaction with the free −SH group of Hcy. These results support the simultaneous binding of Zn2+ and Hcy through the carboxylate oxygen of IDCA@AgNPs. For the further confirmation of binding with Zn2+ and Hcy simultaneously, UV–vis spectra were obtained by mixing Zn2+ (200 μM) and Hcy (2 μM) solutions onto IDCA@AgNPs, and the results are shown in Figure S10 (Supporting Information). As evident from Figure S10, two peaks at around 485 nm (for Zn2+) and around 512 nm (for Hcy) are found as well as the peak of IDCA@AgNPs at 397 nm.

Scheme 2

Proposed Schematic Illustration for the Simultaneous Detection of Zn2+ and Hcy Based on the Aggregation of IDCA@AgNPs

Application of IDCA@AgNPs for Lake Water Samples and Human Urine

To confirm the practical application of IDCA@AgNPs, water samples were collected from the lakes located in Tempe, Arizona, USA. All water samples were filtered through a 0.2 μm membrane and then spiked with different amounts of Zn2+. In the case of Hcy, human urine samples were collected and filtered through a 0.2 μm membrane and then spiked with different amounts of Hcy. A calibration curve of IDCA@AgNPs SPR shifts in the presence of different Zn2+ and Hcy concentrations was prepared. The analytical results are shown in Table S2. The results obtained with IDCA@AgNPs were in good agreement with those obtained using the inductively coupled plasma mass spectrometry (ICP–MS) method, with the relative error of less than 2 and 3% for Zn2+ and Hcy, respectively. These results indicate that the designed probe is applicable for Zn2+ and Hcy detection in water samples.

Conclusions

In summary, a simple, selective, cost-effective, and rapid colorimetric assay for the simultaneous detection of Zn2+ and Hcy using IDCA@AgNPs has been explored in this work. Two analytes, Zn2+ and Hcy, of a solution, could be monitored by the color change of the IDCA@AgNP probe simultaneously. The reported probe (IDCA@AgNPs) showed good selectivity for Zn2+ and Hcy over other several metals ions and amino acids, respectively. Zn2+ and Hcy successfully induced the aggregation of IDCA@AgNPs via complex formation between the IDCA@AgNPs of free carboxylate group of IDCA and Zn2+ and Hcy, yielding a red shift in the SPR peak from 397 to 485 nm (for Zn2+ ion) and 397 to 512 nm (for Hcy), accompanied by the color change from yellow to red. Zn2+ and Hcy have been detected in the wide ranges of pH (pH 3–10). The prepared probe (IDCA@AgNPs) showed good analytical application of environmental water samples, with the relative errors of 2 and 3% for Zn2+ and Hcy, respectively..