Ammonia hydroxide

Absolute ethanol (C₂H₅OH, purity ≥ 99.8%), copper (II) sulfate pentahydrate (CuSO₄·5H₂O, purity ≥ 98.0%), ascorbic acid (C₆H₈O₆, purity ≥ 99.7%), and ammonia hydroxide (NH₃·H₂O, 35%) were purchased from Fisher Chemical (Pittsburgh, PA, USA) and used without further purification. Polyvinylpyrrolidone (PVP, purity ≥ 99.0%), ethylene glycol (C₂H₄(OH)₂, purity ≥ 99.5%), formic acid (CH₂O₂, purity ≥ 95.0%), and sodium perchlorate (NaClO₄, purity ≥ 99.0%) were obtained from Acros Organics (Geel, Belgium). Commercial wetting agent (type 2230) was used in this study, supplied by Marvel Chemical Co. (Taipei, Taiwan). PET ((C₁₀H₈O₄)_n) was purchased from Hsin Yun Co. (Taipei, Taiwan). All chemicals were analytical grade and used as received.

Acetic acid (purity ≧ 99.83%), ethanol (purity ≧ 99.8%), ammonia hydroxide (purity ≧ 35%), and sodium hydroxide (purity ≧ 97%) were purchased from Fisher Chemical (Pittsburgh, PA., USA). Sodium chloride (purity > 99.5%), Congo red (CR), and crystal violet (CV) were purchased from Acros Organics (Geel, Belgium). Alginic acid sodium salt powder, chitosan, reactive blue 4 (RB4), and neutral red (NR) were supplied by Sigma-Aldrich (Natick, MA., USA). Hydrochloric acid (purity > 37%) was obtained from Scharlau (Senmanat, Spain). Calcium chloride (purity ≧ 89%) was purchased from Avantor (Allentown, PA., USA). All chemicals were analytical grade and used without further purification. Agarwood (Aquilaria agallocha Roxb) fruit was obtained from the local farmer in Kaohsiung, Taiwan.

Silica nanospheres (SiO_x NSs) were prepared through a sol-gel process relying on a controlled hydrolysis and condensation of tetraethyl orthosilicate (TEOS, 98%, Sigma-Aldrich) (Green et al., 2003 (link)). An appropriate amount (1.7 mL) of TEOS was added to a solution containing 29.1 mL of absolute ethanol (Pharmco-Aaper), 3.21 mL of deionized (DI) water, and 1.96 mL of ammonia hydroxide (28–30 wt.% in water, Fisher Scientific). The reaction proceeded for 2 h at a stirring rate of 600 rpm to complete the growth of SiO_x NSs. Changing the amount of water and TEOS could tune the size of the synthesized SiO_x NSs. The resultant SiO_x NSs were collected through two cycles of centrifugation and washing with ethanol, and then dried overnight in an oven set at 60°C.

The metabolite profiles of serum were detected by using an LC-MS/MS system consisting of an ultra-high-performance liquid chromatography (UPLC) system (Vanquish, Thermo Fisher Scientific, Waltham, MA, USA) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled to a Q Exactive (QE) HFX mass spectrometer (Orbitrap MS, Thermo Fisher Scientific, Waltham, MA, USA). The UPLC system was used for the separation of biochemicals via the UPLC BEH Amide column. The mobile phase consisted of 25 mmol/L ammonium acetate (Sigma-Aldrich, Darmstadt, Germany) and 25 mmol/L ammonia hydroxide (Fisher Chemical, Waltham, MA, USA) in water (pH = 9.75) (A phase) and acetonitrile (B phase). The autosampler temperature was 4°C, and the injection volume was 3 μl. The QE HFX mass spectrometer was applied to obtain MS/MS spectra in information-dependent acquisition (IDA) mode under the control of the acquisition software (Xcalibur, Thermo Fisher Scientific, Waltham, MA, USA). In this mode, the acquisition software continuously evaluates the full-scan MS spectrum. The electrospray ion (ESI) conditions were as follows: sheath gas flow rate of 30 Arb, auxiliary gas flow rate of 25 Arb, capillary temperature of 350°C, full MS resolution of 60,000, MS/MS resolution of 7500, collision energy of 10/30/60 in negative ion mode (NCE), and spray voltage of 3.6 kV (positive) or −3.2 kV (negative).