Synthesis of Prussian Blue Nanoparticles

PBNPs were synthesized using a one-pot scheme as previously described [9 (link),35 (link),41 (link),44 (link)]. Briefly, 20 mL of 10 mM aqueous FeCl₃•6H₂O (54.06 mg) (Millipore Sigma, Darmstadt, Germany) containing 5 mmol of citric acid (961 mg) (Millipore Sigma, Darmstadt, Germany) was added to an equal volume of 10 mM aqueous K₄[Fe(CN)₆]•3H₂O (84.5 mg) (Millipore Sigma, Darmstadt, Germany) containing 5 mmol of citric acid, under vigorous stirring at 60 °C using a magnetic stirring hot plate. After heating and stirring for 60 s, the solution was allowed to cool to room temperature (RT) for 5 min under constant stirring. The 40 mL solution was then divided equally between two 50 mL tubes and PBNPs were then collected by adding equal volume of acetone (Millipore Sigma, Darmstadt, Germany) and 5 mL 5 M NaCl (Millipore Sigma, Darmstadt, Germany). The solutions were then centrifuged at 10,000 rpm for 15 min at RT. The centrifugation step with 20 mL MilliQ water, 20 mL acetone, and 5 mL 5M NaCl was repeated twice. The final PBNP pellet was sonicated (at 40% amplitude for 30 s using a microtip probe) in 10 mL MilliQ water to achieve colloidal resuspension. The PBNPs were stored under ambient conditions in DI water prior to further bioconjugation.