Silver nitrate

AuNPs in citrate buffer solutions (average core diameter: 20 (18–22), 40 (37–43), 60 (57–63) and 80 (77–83) nm), AgNPs in citrate buffer solutions (average core diameter: 20 (16–24), 40 (36–44) and 60 (52–68) nm and 20 (18–22) nm) AuNPs in 0.1 mM phosphate buffered saline (PBS) (Sigma-Aldrich, Saint Louis, MO, USA) were used, all of them 0.02 mg/mL. Hydrochloric acid (37% w/w) and concentrated acetic acid were supplied by Sigma-Aldrich and VWR Chemicals (Radnor, PA, USA), respectively.
The liquid carrier for AF4 was prepared with sodium azide 0.02% (NaN3, Panreac, Castellar del Vallés, Barcelona, Spain) and pH was adjusted using 0.1 M sodium hydroxide (NaOH, Panreac). Methanol (VWR) was used for cleaning AF4 system.
3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma-Aldrich) and sodium acetate anhydrous (Panreac) were also used. Moreover, it was used silver nitrate (VWR) and gold (III) chloride trihydrate (Sigma-Aldrich).
The water for all the experiments was purified through a Barnstead Nanopure II system.

For the generation of AgNPs, the following reagents were used: sodium hydroxide (Scharlau, Spain), tetradecyltrimethylammonium chloride (≥98) (C14TAC), cetyltrimethylammonium chloride (≥98) (CTAC, Sigma), cetyltrimethylammonium bromide (CTAB) (≥98 %, Sigma), tetrabutylammonium bromide (Bu4B) (≥98 %, Sigma), silver nitrate (%, VWR Chemicals), and Trolox (97%) and chlorogenic acid (>95%) from Sigma-Aldrich, Germany. For the preparation of the mobile phase, ammonium acetate (>98%, Sigma-Aldrich, Germany), sodium dodecylsulfate (>85%) and sodium thiosulfate, from Merck, Germany, were employed.

All chemicals were reagent grade and used without further purification. Copper sulfate (0.1 M), sodium phosphate monobasic monohydrate (NaH₂PO₄.H₂O), sodium phosphate dibasic heptahydrate (Na₂HPO₄.7H₂O), sodium thioglycolate, Phosphate-Buffered Saline tablets (PBS) were purchased from Sigma-Aldrich. Sodium thiosulfate solution (0.1 M) was purchased from Merck and ethylenediaminetetraacetic acid solution (EDTA) 0.1 M and silver nitrate (0.01 M) from VWR.
All experimental solutions were prepared using Milli-Q water (18 MΩ, pure lab chorus 1). Sodium phosphate buffer (PB, 1 mM) contained NaH₂PO₄.H₂O and Na₂HPO₄.7H₂O mixed at concentrations of 0.93 mM and 0.07 mM for pH 6, 0.58 mM and 0.42 mM for pH 7 and 0.12 mM and 0.88 mM for pH 8, respectively. Solutions were autoclaved at 121 °C for 20 min then stored at 4 °C until further use. When needed, further adjustment of pH was conducted using stock solution NaH₂PO₄.H₂O (100 mM) as a base and Na₂HPO₄.7H₂O (100 mM) as an acid. Silver neutralizing solution was prepared on the experimental day as described by Butkus et al. (2004) (link). The solution contained 12 g/l of sodium thioglycolate and 0.1 M/l of sodium thiosulfate.