Nitric acid

Tissue samples (including brain, gill, gut, kidney, liver, gonad and muscle) were collected by necropsy to determine the total Se concentration. Tissues were weighed (mg dry weight) and transferred into 50 mL digestion tubes containing 1 mL 70% nitric acid (metal grade, Sigma, Burlington, MA, USA). Digestion was carried out in heat block for 2 h at 95 °C and 0.25 mL 30% H₂O₂ (Sigma, Burlington, MA, USA) to the tube and continued to be heated for another 2 h at 95 °C. The digested mixture then was diluted four-fold with 5% nitric acid and filtered through 0.45-µm Hydrophilic Teflon filters (Sigma, Burlington, MA, USA). Filtered samples were furthered diluted five-fold with 5% nitric acid in 15-mL Falcon tubes to achieve a final volume of 5 mL. Indium was added as an internal standard for determination of consistency of machine efficiency. ICP-MS (Agilent 7500, Santa Clara, CA, USA) was used to determine the total Se concentration. Using spiked samples and certified reference materials (DORM-4, National Research Council Canada, Ottawa, ONT, Canada), the average Se recovery of our method was 92.4%. MilliQ water was used as blank sample.

Tetrachloroauric acid trihydrate (99.99%), sodium citrate dihydrate (≥ 99%), Triton X-100 (laboratory grade), sodium borohydride (≥ 98%), silver nitrate (> 99%), poly(ethylene glycol) methyl ether thiol (mw 2000), poly(allylamine) hydrochloride (PAH, mw 50000), sodium poly(4-styrene sulfonate) (PSS, mw 70000), nitric acid (≥ 65%), nitric acid (1M), hydrochloric acid (≥ 37%), sulfuric acid (95.0–97.0%), hydrogen peroxyde (30% w/w), ethanol (≥ 99.8%), 2-propanol (99.5%), and ethyl cellulose (10 cP; 22 cP; 46 cP; 100 cP) were bought from Sigma Aldrich (Milano, Italy); α-mercapto,ω-carboxy poly(ethylene glycol) (mw 3000), poly(ethylene glycol) methyl ether thiol (mw 5000), poly(ethylene glycol) methyl ether thiol (mw 10000), and poly(ethylene glycol) methyl ether thiol (mw 20000) were bought RAPP Polymere (Tübingen, Germany); Ethylene glycol (99.5%) and ammonia solution in water (30% w/w) were bought from Carlo Erba Reagenti S.p.A. (Milano, Italy) Glass coverslides (22 × 26 mm, 0.14 mm thickness) were bought from Delchimica Scientific Gòassware (Napoli, Italy).

As an EPR sample, we prepared
a Copper II ions (a typical reference EPR sample already used in with
NV centers^{35 (link)}). We prepared a nitric acid
solution by diluting concentrated nitric acid (90%, Sigma-Aldrich,
695041) in Milli-Q water. The sample itself was prepared by dissolving
copper sulfate powder (Merck, 2790) in nitric acid solution to favor
the formation of copper II hexaaqua Cu(H2O)₆ complex ions,
which have a well-known EPR spectrum.^{36 (link)} The final concentration of the solution is approximately 1 μm.
The sample was pumped through a Y shaped microfluidic channel (0.1
mm width, 0.5 mm thick) on a PDMS chip with a syringe.

Fresh mouse brain tissues were digested with 500 μL nitric acid (Sigma) at 100 °C for 30 min. After the samples returned to room temperature, they were diluted 20 times with 0.1% nitric acid (Sigma). Then, the samples were measured using AAS (ZEEnit700P, Analytikjena, Germany) as previously described [58 ].

SiO₂/CGA and SiO₂/PEG/CGA organic–inorganic hybrids materials were synthesized by the sol-gel method according to a procedure reported in a recent study [34 (link)]. A solution of tetraethyl orthosilicate (TEOS, reagent grade, 98%, Sigma Aldrich, Steinheim am Albuch, Germany) was used as a precursor of the SiO₂ inorganic matrix. TEOS, ethanol 99% (Sigma Aldrich, Steinheim am Albuch, Germany), nitric acid (solution 65%, Sigma Aldrich, Steinheim am Albuch, Germany), and water were added in a solution under stirring; nitric acid was used to promote the kinetics of hydrolysis and condensation reactions. The molar ratios among the reagents in the obtained solution are: EtOH/TEOS = 6.2, TEOS/HNO₃ = 1.7, H₂O/TEOS = 6.
The hybrid materials were obtained using ethanol solutions of CGA at different percentages (5 wt %, 10 wt %, 15 wt %, 20 wt %), while a high amount of polyethylene glycol (MW = 400, Sigma Aldrich) (PEG 50 wt %) dissolved in ethanol was added to the pure silica matrix (to reach 50 wt %) before preparing the SiO₂/PEG/CGA materials. The different prepared solutions were put at room temperature until the gel was obtained, and then left into an oven at 40°C to allow for the removal of the residue solvent, thus avoiding the thermal degradation of the drug. The flow chart of the sol-gel process is shown in Figure 1.