3.1. Phenylalanine and gold reaction
Phe as an amino acid carries amine and carboxyl groups which can act as chelating ligands to coordinate with metals such as Au (III) 32. When Phe and HAuCl4 are mixed, the ligand exchange takes place in a few minutes. The electronic transitions causing the absorption bands of HAuCl4 in UV region (220 nm and 293 nm) are assigned to the ligand-to-metal charge transfer from p orbital of Cl to d of Au 33. The absorbances related to these transitions are drastically decreased by adding Phe, indicating a ligand exchange (Fig. S1).
All 20 natural amino acids including Phe are able to reduce Au (III) ions to Au (0) in an appropriate pH and temperature 34,35, or by the aid of irradiation 36. However, the empirically elucidated mechanisms are limited to few amino acids including glycine 37, alanine 38, tryptophan 39, and dopamine 40 (the decarboxylated derivative of 3,4 dihydroxyphenylalanine (L-DOPA)). In the case of tryptophan or L-DOPA, the functional groups of the side chains are responsible for the reduction. However, in the case of glycine and alanine the common features of amino acids, alpha amine and carboxyl moieties are involved in gold (III) reduction.
When a molecule is oxidized, electron is removed from the highest occupied molecular orbital (HOMO). Therefore, the oxidation potential of a molecule, as an indicator of its tendency to lose electron, is correlated with its HOMO energy level 35,41,42. The calculated HOMO energies for alanine and Phe are similar, and the ionization of Phe is assigned to the joint contribution of the nitrogen lone pair and the π orbitals from the phenyl group 43. On the other hand ,the electronic structure of Phe can be considered as the sum of benzene and alanine 44. Therefore, the reaction pathway of gold reduction by alanine seems to be valid for Phe (Fig. 1). With valid assumption of this pathway, Phe can produce two atoms of gold in two steps. Phenyl pyruvate (PhePyr) and NH4+ are produced at the first step by removing the amine from Phe, and in the second step, PhePyr is oxidized to phenylacetatic acid (PheAc) by production of CO2.
The proposed pathway was evaluated by the production of NH4+, removing of amine from Phe, and the production of PhePyr and PheAc. However, prior to evaluating the proposed pathway, the following five observations were taken into account to adjust the reaction condition tactfully.
First, the hydrothermal gold (III) reduction takes place at temperatures higher than 60°C and pH values ≥ 7 (Fig S2). Therefore, the synthesis reaction must be carried out in a temperature and pH range in which the hydrothermal reduction does not occur. This is important from stoichiometric point to make sure that all reduction electrons come from the reducing agent and not water.
Second, all pH values were adjusted by NaOH or HCl, and we avoided using any kind of buffer to adjust the pH of Phe and gold reaction. For example, phosphate buffer (PB) is usually mentioned as a non-involving buffer in gold reduction, however, we have observed that the reduction of gold happens in high concentrations of sodium PB. As depicted in Fig. S3, PB (1 M) could produce precipitates at temperatures from 40°C to 70°C and pHs of 5, 6, and 7. However, at pH = 8 no obvious particle formation was detected. In addition, by applying lower concentration of 0.1 M PB, the precipitates were observed only in pH = 5 and temperatures from 50°C to 70°C. This indicates the contribution of PB in complexation as well as the reduction of gold ions.
Third, the reduction by PhePyr is a fast reaction, and produces PheAc. As depicted in Fig S4, at room temperature, the reduction happens in pHs ≥ 7. As temperature rises, the reduction was observed in pHs ≤ 7 too. PhePyr is capable of reducing gold ions at pH = 6 and 7 at 60°C, and there is not any obstacle in front of the second step of the proposed pathway to proceed. The FTIR spectra of air-dried supernatant of the reaction production well-resembles PheAc.
Forth, as it is indicated in Fig. S5, PheAc does not go further oxidation, and hence, it is supposed to be the finial oxidized product especially if the pH of the reaction is adjusted below 7 (Fig. S4).
Fifth, unlike PhePyr, the reduction by Phe does not happen immediately at room temperature, and it roughly takes one to two days to produce visible particles. Hence, the activation energy for gaining electrons from amine seems to be greater than that from aldehyde (Fig. S6).
Taking these observations into account, the reaction mixture of Phe and HAuCl4 was setup to be exempted from any additional buffering agents, and the pH value was adjusted to 6 by NaOH or HCl, and the reaction temperature was set to 60°C. In this condition, no thermal reduction takes place, and Phe and PhePyr are able to reduce gold ions while PheAc is not. As the reaction by PhePyr is faster than that by Phe, there is no kinetic bottleneck to cause PhePyr to accumulate. Therefore, it is expected that the final oxidized product is PheAc when the molar ratio of Au:Phe > 2. However, as it is going to be explained, proceeding the second step of the reduction reaction is determined by initial pH value.
In the first step of the reduction reaction, atomic gold and ammonium are produced through the conversion of Phe to PhePyr. The ammonium production was confirmed by Nessler’s reaction. To assure that the other reagents as Phe and HAuCl4 do not interfere with the detection of ammonium, their interaction with Nessler’s reagent were examined too. The reaction of Nessler’s reagent with various concentrations of Phe, ammonium and HAuCl4 are indicated in Fig. S7. As expected, Phe did not induce any color change, whilst ammonium generated a color shift from transparent to orange with a proper linear relation in the range of 1.5 to 50 mM. Nessler’s reagent reduces HAuCl4, and produces precipitates. These precipitates do not interfere with the detection of ammonium, because they can be easily removed by spinning down to leave the supernatant clear. As depicted in Fig. 2A, ammonium production was confirmed in all reactions of various molar ratios of Au:Phe (0.5, 1, 2.5, and 3). The evaluated ammonium concentrations were equal to the initial concentrations of Phe in the cases of Au:Phe = 2.5 and 3, showing the conversion of all amines of Phe to ammonium. Benzoquinone reacts with amine functional group selectively (Fig. S8). Figure 2B indicates the removal of amine from Phe in the reduction reaction. No amine was detected in the samples of Au:Phe = 2.5, and 3, showing that all Phe molecules have lost their amine group.
The second step of the reduction reaction is validated by FTIR spectroscopy by showing the production of PheAc. As Phe, PhePyr, and PheAc hold similar functional groups, we do not discuss the spectra in detail or try to assign the absorbances to specific molecular vibrational modes which are well-explained elsewhere 45–47. Instead, we have used the similarity of the spectra as fingerprints to show the production of PheAc. Considering the stoichiometry of the proposed pathway, the final oxidized product must be totally PheAc when Au:Phe molar ratio is greater than two (here, 2.2). As shown in Fig. 3F, the spectra of Phe and gold reaction well-resembles PheAc when Au:Phe = 2.2 and initial pH is 7. This verifies the proposed pathway for reduction of gold ions by Phe.
Besides being the proof for the pathway, the obtained results by FTIR are notable especially for adjusting the reaction condition. As indicated in Fig. S9, the microscopic shapes of the air-dried Phe, PhePyr, and PheAc are very different that indicates the involvement of distinct molecular forces driving the self-assembling process. Different functional groups drive different combinations of hydrogen bonding which is an important force in the self-assembling process of similar molecules 48,49. The building blocks of AuNC-carrying self-assembled structure as sensor are produced through the reduction reaction that may influence the features of the sensor.
When the molar ratio of Au:Phe is 0.5, the self-assembled structure is mainly composed of Phe (Fig. 3A&B). While, when Au:Phe > 2 and initial pH = 6, the second step of the reduction by PhePyr is restricted, and PhePyr accumulates (Fig. 3C&D). The accumulation of PhePyr shows that the second step of the reduction does not proceed to produce PheAc. As previously indicated (Fig S4), the reduction of gold ions by PhePyr is faster than that of Phe (Fig S4, S6), and the accumulation of PhePyr is not expected through a kinetic barrier. Also, the reduction of gold by PhePyr takes place at pHs ≥ 4 at 60°C (Fig. S4), and pH does not drop drastically to an unsuitable range when Phe and HAuCl4 reacts. Moreover, the presence of PheAc in the reaction mixture of PhePyr and HAuCl4 does not prevent reduction by PhePyr (data not shown). Hence, considering these observations the accumulation of PhePyr was not expected, and we could not explain it documentarily. A probable explanation may be that the pH modulates the tendency of PhePyr to self-assemble or react further with HAuCl4 which both are directed with its functional groups. Whatever the reason is, its consequence is important; The building blocks are totally made up of PhePyr in the case of Au:Phe > 2, and initial pH = 6. Also, extra gold ions remain in the solution due to an in-complete reaction. Increasing the fluorescence intensity of such a solution by adding NaBH4 confirms the presence of extra gold ions (data not shown). On the other hand, when Au:Phe > 2, and initial pH = 7 the two steps of reduction reaction take place, and PheAc is produced (Fig. 3E&F), by which no fluorescent NC could form.