The sensing model in the solutions

In this section, we demonstrated the optimum condition for sensing Hg²⁺ by using a functionalized-AuNP probe. The diameter of 13 nm AuNPs, with numerous citrate ions attached on the surface, had high stability for the synthesis of AuNP probes by chemical modification. The AuNP probe was developed with 3-mercaptopropionic acid (MPA) self-assembled upon the surface by Au-S covalent bond to MPA/AuNPs and the probe exposed carboxylic groups exteriorly. The MPA/AuNPs were prepared in order to sensor Hg²⁺ due to the recognition of ions-induced aggregation of AuNPs with the carboxylic groups. The process was coupling interaction to bring AuNPs closer approach to form lager particles that exhibited broadening of surface plasmon absorption band accompanied with the color changed from wine-red to purple. In Figure 4.4, the UV-vis spectrum revealed the results of 10 μL Hg²⁺ standard solution (10 mM) added into 1.5 mL MPA/AuNPs (7.5 nM) in tris-borate buffer (pH 9.0). The dispersive MPA/AuNPs showed the maximum absorbance at 518 nm; upon Hg²⁺ ion presenting, the changed color that displayed the red-shift signal from 518 nm to 650 nm indicated the occurrence of aggregation. In this study, we took the advantage of the color change called colorimeter and examined the phenomenon even by naked eye in the aggregation-induced processing.

After the addition of Hg²⁺, the MPA/AuNPs were promoted to aggregate with time.

From our observation in Figure 4.5, the absorbance at 518 nm decreased gradually while the absorbance at 650 nm increased during the process that indicated the dispersive particles aggregate into larger particles. Therefore, we defined the AuNPs with maximum absorbance at518 nm (A518) and 650 nm (A650) as dispersive and

aggregated condition respectively. As a result, the ratio of A650 to A518 (A650/A518) was raised when the AuNPs proceeding aggregation and then kept a constant until reacting to equilibrium.

Figure 4.4 UV-Vis absorbance spectra that (A) the dispersive MPA/AuNPs and (B) aggregated MPA/AuNPs in the presence Hg²⁺ displayed the red-shift signals from 518 nm to 650 nm. And the picture showed below.

Figure 4.5 UV-Vis spectra illustrated the absorbance of vicissitudes MPA/AuNPs aggregated after Hg²⁺

ions addition with time interval.

To demonstrate the sensing ability of MPA/AuNPs, we prepared MPA/AuNPs solution mixed with 50 mM tris-borate buffer (pH 9.0) to detect the Hg²⁺ concentration.

After addition of the Hg²⁺ standard solution (100 μL) into the MPA/AuNPs (900 μL;

2.5 nM), the result showed in Figure 4.6: the A650/A518 ratios increased with the proper concentration of Hg²⁺.

After the incubation of 10 minutes, we observed that the ratios increased proportional to the amount of Hg²⁺ due to the different degree of aggregation in the range from 4 μM to 8 μM. The ratios reached a constant, which indicated that all dispersive AuNPs transformed aggregation until augmenting concentration of Hg²⁺ up to 8 μM. With comparison of the same condition under various incubation times, the aggregation proceeded continuously and the A650/A518 ratios became larger after 30 minutes. But the detection limit still remained at 4 μM in the interval of 30 minutes.

After 24 hours of incubation, we monitored that the detection limit of Hg²⁺ improved to 3 μM in Figure 4.7. With the low concentration of Hg²⁺, it took longer time to clearly monitor the A650/A518 ratios increasing accompanied with the color changed from wine-red to purple. Herein, the incubation time is one of the factors to affect the sensor ability in the MPA/AuNPs sensor system.

Figure 4.6 (A) The A650/A518 responses of MPA/AuNPs after addition of various concentrations of Hg²⁺

ions (1 μM ~ 10 μM) with various incubation time (10 minutes, 20 minutes, 30 minutes). (B) The samples that changed the colors at the titrating different Hg²⁺ concentrations.

Figure 4.7 The A650/A518 responses of MPA/AuNPs after addition of various concentrations of Hg²⁺ ions (1 μM ~ 10 μM) compared with 30 minutes and 24 hours of incubation.

The MPA molecules without binding to AuNPs were considered as free ligands.

The excess MPA molecules would influence the detection of Hg²⁺ in the reaction.

Consequently, we treated MPA/AuNPs solution with centrifugation at the speed of 15000 rpm for 15 minutes to get rid of the free MPA molecules. The purified MPA/AuNPs solution improved the sensitivity under the lower Hg²⁺ concentration.

The effect revealed that the free MPA molecules exposed both its mercapto and carboxylic groups that can capture Hg²⁺ to influence the sensitivity in MPA/AuNPs system. As a result, we could get the better detection limit for sensing Hg²⁺ at 3 μM after centrifugation in Figure 4.8.

Figure 4.8 The A650/A518 responses of MPA/AuNPs after centrifugation with various incubation time (10 minutes, 20 minutes, 30 minutes).

Furthermore, we investigated the relationship between the concentration of MPA/AuNPs and the probing Hg²⁺. We prepared two concentrations of MPA/AuNPs (2.5 nM and 7.5 nM) to detect Hg²⁺. The result showed that 7.5 nM MPA/AuNPs aggregated at 12 μM of Hg²⁺ in the reaction time of 30 minutes, and lower concentration of MPA/AuNPs aggregated at 4 μM in Figure 4.9. Besides, we also used the purified MPA/AuNPs by centrifugation to observe the sensitivity in 2.5 nM and

7.5 nM MPA/AuNPs systems (Figure 4.10). The dilute MPA/AuNPs maintained better sensitivity toward Hg²⁺ with comparison of the high concentration of MPA/AuNPs. To obtain sensitive MPA/AuNPs probe to detect Hg²⁺, it’s necessary to dilute the MPA/AuNPs solution as possible to acquire lowest detection limit. However, decreasing of MPA/AuNPs concentration caused small absorption and showed high transparent which were not available for observation by naked eye.

Figure 4.9 Two concentrations of MPA/AuNPs showed the A650/A518 responses of MPA/AuNPs in different range of Hg²⁺

Figure 4.10 Two concentrations of MPA/AuNPs showed A650/A518 responses of MPA/AuNPs in different range of Hg²⁺ ions after centrifugation.