Maturing conditions of bimetallic nanocomposites as a new factor influencing Au–Ag synergism and impact of Cu(II) and/or Fe(III) on luminescence

Gold–silver synergism in luminescent nanoclusters has been widely studied, typically by altering Au:Ag ratios. Here, we systematically investigated the effect of maturing conditions while keeping the ratio constant (5:1). Reference systems of gold nanoclusters and proteins were also examined.

Maturing conditions significantly influenced fluorescence and synergism, with the strongest effect observed at 37 °C for 2.5 h. Luminescence stability was further tested in the presence of Cu(II) and Fe(III) ions, which caused varying extents of quenching. These findings reveal maturing as a key factor controlling Au–Ag synergism and nanocluster luminescence.

The original article

Maturing conditions of bimetallic nanocomposites as a new factor influencing Au–Ag synergism and impact of Cu(II) and/or Fe(III) on luminescence

Veronika Svačinová, Tomáš Pluháček, Martin Petr and Karolina Siskova

R. Soc. Open Sci.12: 241385

https://doi.org/10.1098/rsos.241385

licensed under CC-BY 4.0

Selected sections from the article follow. Formats and hyperlinks were adapted from the original.

In the present work, we show that besides varying the Au : Ag ratio in the reaction mixture (as known from the literature), there is another possibility for tuning Au–Ag synergism while keeping the same molar ratio of Au : Ag in the reaction mixture. We introduce a new parameter and call it maturing conditions. The maturing conditions of GSNCs involved either time variations (0 h, 1.25 h, 2.5 h) at room temperature or elevated temperatures (37°C, 50°C) kept for 2.5 h. Indeed, we discovered that the time of sample maturing at room temperature (i.e. the delay between the sample synthesis and its purification by dialysis), possessing otherwise the same composition (i.e. reactant concentrations, their same molar ratios), being mixed exactly in the same way and under the same conditions, has a tremendous impact on their final luminescent features. When we compared our GSNCs to the identically synthesized and matured AuNCs entrapped in BSA and determined their FQY (which is more accurate than the direct comparison of fluorescence emission intensity, especially when the fluorescence maximum shifts and the width of fluorescence band changes), it turned out that the Au–Ag synergism can be achieved in specific cases exclusively.

Furthermore, inspired by the biological origin and function of the used protein, we have also investigated the influence of physiological (37°C) versus elevated temperature (50°C) in the direct comparison to room temperature maturing in the process of prolonged sample maturing (2.5 h). While GSNCs matured at 37°C for 2.5 h provided the best Au–Ag synergism, elevated temperature during sample maturing (50°C) led rather to changes of the protein secondary structure (determined by circular dichroism) and virtually no Au–Ag synergistic effect was observed. The results reveal that Au–Ag synergism can be finely tuned by setting appropriate maturing conditions of bimetallic nanoclusters embedded in the protein scaffold. Maturing conditions thus represent a new factor in the viewpoint of Au–Ag synergism.

For the most prominent GSNCs, revealing the best synergism, it is demonstrated that the fluorescence of GSNCs can be quenched more efficiently by an excess of Cu(II) rather than Fe(III) ions. The exact mechanism is currently unknown, but a plausible reason is suggested by us and further investigations are envisaged. Since both selected metal ions are crucial in various cellular metabolic pathways and their impaired metabolism related to several neurodegenerative diseases and/or cancer [27], their impact on GSNC represents an important issue.

2. Results and discussion

2.2. Characterization of the samples and evaluation of Au–Ag synergism

2.2.1. Metallic part characterization (by ICP-MS, XPS, UV–visible absorption)

Theoretical and experimental concentrations (in mg ml⁻¹) of Au and Ag in each metallic sample (GSNC and AuNC types) are summarized in electronic supplementary material, table SI-1; while the molar ratio of Au : Ag (theoretical and experimental) for bimetallic GSNC samples is calculated in electronic supplementary material, table SI-2. It should be noted that the experimental values of Au and Ag concentrations were accurately determined by ICP-MS. Interestingly, the experimental values of Au contents slightly increased from 368 ± 4 to 381 ± 6 µg ml⁻¹ with prolonged time of maturing applied at room temperature (electronic supplementary material, table SI-1). It thus corroborated the assumption that prolonged time may have an effect on metallic ion incorporation into the protein scaffold. Obviously, it is valid only for Au(III), while not for Ag(I) because the experimental content of Ag is around 40 ± 3 µg ml⁻¹ in all types of GSNC samples (electronic supplementary material, table SI-1). Differences in Ag incorporation between individual GSNC samples are only negligible; they lie within the experimental error. The GSNC samples matured at elevated temperature (50°C) for prolonged time (2.5 h) did not cause any significant increase of Au content in comparison to the samples matured at room temperature for the same period (electronic supplementary material, table SI-1). On the other hand, usage of the physiological temperature of maturing led to Au(III) content slight decreasing (but within 8%). Based on the experimental concentration values of both metals, Au and Ag, the percentage yield of each metal within a particular metallic (GSNCs as well as AuNCs) sample can be calculated, the values being listed in table 1.

To determine oxidation states of Au and Ag within our metallic samples, XPS measurement has been employed. The XPS signals of GSNC samples are shown in figure 2, while those of AuNC samples in electronic supplementary material, figure SI-1.

R. Soc. Open Sci. 12: 241385: Figure 2. XPS signals in Au and Ag characteristic regions recorded for our representative metallic samples: (A) Au 4f region of GSNC 0 h RT, (B) Ag 3d region of GSNC 0 h RT, (C) Au 4f region of GSNC 1.25 h RT, (D) Ag 3d region of GSNC 1.25 h RT, (E) Au 4f region of GSNC 2.5 h RT, (F) Au 4f region of GSNC 2.5 h 37°C, (G) Ag 3d region of GSNC 37°C, and (H) Au 4f region of GSNC 2.5 h 50°C.

2.2.3. Luminescent properties of gold–silver nanoclusters and extent of Au–Ag synergism

Fluorescence emission was measured using the same excitation wavelength (460 nm) to investigate luminescent properties of GSNC samples generated under different maturing conditions. Normalized spectra are depicted in figure 4.

R. Soc. Open Sci. 12: 241385: Figure 4. Fluorescence emission of GSNC samples matured under different conditions: 0 h RT (green curve), 1.25 h RT (black curve), 2.5 h RT (brown curve), 2.5 h 37°C (blue curve) and 2.5 h 50°C (red curve). Excitation was at 460 nm in all five cases. Normalized spectra are shown for the sake of a clear presentation of intensity maximum shifts and emission band broadening.

The position of emission maximum of GSNC 0 h RT sample is located at 615 nm (figure 4). This corresponds very well to the position of fluorescence emission maxima reported for Au : Ag systems (also 5 : 1 ratio) prepared by Zhang et al., albeit they exploited peptide (GSH) as the template for nanocluster synthesis [6]. While prolonging the maturing time, the emission maximum red-shifts to 625 nm and further to 640 nm (figure 4) as obvious for GSNC 1.25 h RT and GSNC 2.5 h RT, respectively. While increasing the maturing temperature, the emission maximum continues to red-shift to 685 nm (GSNC 2.5 h 50°C). Similarly, the emission maxima of our monometallic AuNC samples red-shifted gradually either by 5 nm (going from samples matured at room temperature for 0 h (655 nm) to that matured for 2.5 h at 37°C (670 nm)), or by 10 nm (for the AuNCs matured at 50°C for 2.5 h) as can be clearly seen in electronic supplementary material, figure SI-4.

3. Conclusions

In this work, maturing conditions representing a new factor that can help to adjust Au–Ag synergism are introduced and investigated. Maturing is defined here as the procedure between the synthesis (finished by MW irradiation) and the dialysis (that stops the nanostructure evolution). It is well documented that this factor has been overlooked, underestimated, not considered or investigated intentionally so far. Au–Ag synergism is evaluated as the ratio of FQY values of GSNCs versus AuNCs. Bimetallic (GSNCs) and monometallic (AuNCs) nanocomposites were synthesized using MW irradiation and exploiting protein as a template to restrict the growth, so that luminescent nanoclusters and not plasmonic particles are formed. The protein treated in the same way (during the synthesis and maturing procedure) as the metallic nanocomposites served as a referent system. The selected variations of maturing conditions included either time (0 h, 1.25 h, 2.5 h) or temperature (room temperature, 37°C, 50°C). It was revealed that maturing time plays a significant role leading to an increase of Au–Ag synergistic effect. Based on the increasing Au–Ag synergism, the GSNC samples can be arranged as follows: 0 h RT < 1.25 h RT < 2.5 h 50°C < 2.5 h RT < 2.5 h 37°C. Regarding nanocluster size increase and PSD broadening, both evidenced by STEM (for metallic parts only) and DLS (hydrodynamic diameter of the whole nanocomposite in aqueous solution), the nanocomposites can be arranged into the order: 0 h RT < 2.5 h RT < 2.5 h 50°C. This order does not correlate with the observed increase of Au–Ag synergistic effect. In fact, a temperature of 50°C held for 2.5 h influences the secondary structure of the protein matrix significantly. Therefore, besides the increasing nanocluster size and PSD broadening during GSNC maturing, other factors, such as changes of the protein secondary structure for instance, play a key role in Au–Ag synergism as well. The results thus imply that maturing conditions of GSNC samples represent a very important factor leading to a further fine tuning of their luminescent properties, hypothetically by the different structural arrangement of atoms within Au–Ag nanoclusters. The stability of the GSNC luminescent signal was further investigated in the presence of Cu(II) and/or Fe(III) because these two selected metal cations are relevant cofactors in living systems. The observed differences in the extent of luminescence quenching caused by Cu(II) and/or Fe(III) in the concentration range of 0−20 mM were discussed and a plausible reason for such a phenomenon was suggested.

4. Methods

4.4. Inductively coupled plasma mass spectrometry

Before ICP-MS analysis, 500 µl GSNC aliquots were dried in a vacuum rotary evaporator to dryness. The total silver and gold levels were accurately determined using a validated ICP-MS method [47]. Briefly, the dried aliquots were digested using a digestion mixture of 2 ml of concentrated nitric acid and 2 ml of concentrated hydrochloric acid in an MLS 1200 mega closed vessel microwave digestion unit (Milestone, Italy) according to the power-controlled digestion programme. The digests were allowed to cool down to laboratory temperature and diluted with ultrapure water in 25 ml volumetric flasks. The total silver and gold levels were acquired by an Agilent 7700x ICP-MS (Agilent Technologies Ltd, Japan) using seven-point external calibration within a concentration of 10–2000 µg l⁻¹ for Ag and 100–10 000 µg l⁻¹ for Au. The quality control sample at a concentration level of 500 µg l⁻¹ for Ag and 5000 µg l⁻¹ for Au was regularly analysed every ten samples to ensure the quality of the acquired results. All ICP-MS measurements were performed in six replicates, and the results are expressed as an average ± s.d.

4.7. X-ray photoelectron spectroscopy

The XPS measurements of most samples were carried out with a PHI 5000 VersaProbe II XPS system (Physical Electronics) with a monochromatic Al-K_α source (15 kV, 50 W) and a photon energy of 1486.7 eV. All the spectra were measured in a vacuum of 1.1 × 10^–7 Pa and at a room temperature of 20°C. Dual beam charge compensation was used for all measurements. The spectra were evaluated with MultiPak software, version 9 (Ulvac-PHI, Inc., Chanhassen, MN, USA).

The sample labelled as GSNC 0 min RT was analysed with a Nexsa G2 XPS system (Thermo Fisher Scientific) also equipped with a monochromatic Al-K_α source and photon energy of 1486.7 eV. The spectral acquisition was maintained at 1.6 × 10^–9 mBar, and at a controlled ambient temperature of 20°C. Charge neutralization techniques were systematically employed throughout the measurement. Spectral analysis and data interpretation were conducted using Avantage software version 6.5.1 provided by Thermo Fisher Scientific.