Sodium dihydrogen phosphate

L, L-APM, L, D-APM, D, L-APM, and D, D-APM were kindly provided by Ajinomoto Co., Inc. (Tokyo, Japan) and used as standards for HPLC analysis. Eleven samples of commercially available L, L-APM (samples 1–11) were obtained from domestic and foreign manufacturers. Acetonitrile and methanol were of HPLC grade and purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Sodium dihydrogen phosphate and disodium hydrogen phosphate were of analytical grade and purchased from Wako Pure Chemical Industries, Ltd. The water used was ultrapure, purified to 18 MΩ·cm using a Milli-Q Gradient A10 (Merck Millipore Corporation, Billerica, MA, USA). All chemicals and samples were weighed using a XS205DU microbalance (Mettler-Toledo International Inc., Columbus, OH, USA).

Membrane protein CD80 was used as the target, and biotin-labelled anti-CD80 antibody was used as the probe molecule (both were provided by Sysmex Corporation)^{39 (link)}. The stock solution of CD80 dispersion was 250 μg/mL and diluted to the target concentration for the experiment using phosphate buffer as the solvent. The used phosphate buffer was self-made by mixing disodium hydrogen phosphate (197-02865, Wako Pure Chemical Industries) and sodium dihydrogen phosphate (197-09705, Wako Pure Chemical Industries) at 10 mM, pH = 7.0.
Then, the anti-CD80 antibody-modified streptavidin-labelled polystyrene latex beads (2 μm in diameter, 24160, Polysciences) were used as probe microparticles. The used phosphate buffer was the same as that used for the CD80 dilution. First, streptavidin-labelled polystyrene particles were diluted to 1.148 × 10⁹ particles/mL with 10 mM phosphate buffer. Biotin-labelled anti-CD80 antibody (undiluted concentration 1 mg/mL) was diluted to 20 μg/mL in 10 mM phosphate buffer. Equal amounts of the prepared Latex bead solution and anti-CD80 solution were mixed, and the mixture was placed in an incubator at 41.7 °C for 1 h. The beads were removed from the incubator and washed five times by centrifugation (10,000×g, 5 min) with 10 mM phosphate buffer. The prepared anti-CD80 antibody-modified beads were stored refrigerated at 4 °C.

Ultrapure water with a specific resistance of 18.2 MΩ/cm was produced in an Arium 611 Ultrapure Water System (Sartorius Co., Goettingen, Germany) and used in our experiments. Methanol (high-performance liquid chromatography [HPLC]-grade), acetonitrile (HPLC-grade), choline chloride, propionic acid, n-butyric acid, ethyl acetate (EtOAc), formic acid and 1-N hydrochloric acid were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). N,N′-dicyclohexylcarbodiimide (DCC) was purchased from Peptide Institute, Inc. (Osaka, Japan); 4-N HCl/dioxane and N,N-dimethylformamide (DMF) were purchased from Watanabe Chemical Industries, Ltd. (Hiroshima, Japan); N,N-dimethyl-4-aminopyridine (DMAP) was purchased from Kokusan Chemical Co., Ltd. (Tokyo, Japan); and ACh chloride, pivalic acid and deuterium oxide were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). (2-aminoethyl)trimethylammonium chloride hydrochloride was purchased from Sigma-Aldrich, Inc. (Ontario, Canada). All other purchased chemicals—including dichloromethane (DCM), sodium dihydrogen phosphate and disodium hydrogen phosphate—were from Fujifilm Wako Pure Chemical Industries, Ltd. (Osaka, Japan). PCh, BCh, LCh and (2-aminoethyl)trimethylammonium pivaloylamide (EN) were synthesized in our laboratory.

FS⁻ (purity: <100%, product no.: 065-00252) and MB⁺ (purity: 98.5+%, product no.: 133-06962) were purchased from FUJIFILM Wako Pure Chemical Corp., Osaka, Japan. The synthesized particles and photofunctional dyes (FS⁻ or MB⁺) were mixed in an agate mortar (mortar diameter and depth: 6.6 and 3.8 cm; rod length and diameter: 8.3 and 2.5 cm) at the weight ratio of 5:1 for the mixing time of 10 min at room temperature. The resulting powder mixture of the particles and dyes was washed with either EtOH, IPA, or ultrapure water (FS⁻ only), and the solid phase was obtained via centrifugation (3 °C, 13,000× g, 10 min). The washing process was repeated until the supernatant solution became clear. However, it was difficult to achieve clarity by washing only with ultrapure water. The washed particles were dried overnight at 60 °C to obtain ASPs and Cl-containing ASPs immobilized with FS⁻ and MB⁺. FS⁻ and MB⁺ in the solution were also prepared for the comparison. In particular, both FS⁻ and MB⁺ powders were dissolved in 1 mM of phosphate buffer at the concentration of 1.0 × 10⁻⁷ M. Here, the buffer was prepared by mixing sodium dihydrogen phosphate (purity: 99+%, product no.: 197-02865) with sodium hydrogen phosphate (purity: 99+%, product no.: 197-09705), which were purchased from FUJIFILM Wako Pure Chemical Corp., Osaka, Japan.

Chitosan (200–600 mPa·s; 0.5% in 0.5% acetic acid at 20 °C, Tokyo Kasei Kogyo, Tokyo, Japan), genipin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), acetic acid (FUJIFILM Wako Pure Chemical Corporation, Japan), disodium hydrogen phosphate (Na₂HPO₄; FUJIFILM Wako Pure Chemical Corporation, Japan), sodium dihydrogen phosphate (NaH₂PO₄; FUJIFILM Wako Pure Chemical Corporation, Japan), Giemsa Stain Solution (Merck, Darmstadt, Germany), 10% formalin neutral buffer solution (FUJIFILM Wako Pure Chemical Corporation, Japan), Dulbecco’s PBS (PBS; Sigma-Aldrich, St. Louis, MO, USA), polyetherurethane (PU) film containing 0.1% zinc diethyldithiocarbamate (ZDEC) (ZDEC-PU; Hatano Research Institute, Hadano, Japan), PU film containing 0.25% zinc dibuthyldithiocarbamate (ZDBC) (ZDBC-PU; Hatano Research Institute, Hadano, Japan), high-density polyethylene sheet (HDPE; Hatano Research Institute, Hadano, Japan), Chinese hamster lung fibroblast V79 cells (V79 cells; RIKEN RCB0008 and Japan Health Sciences Foundation JCRB0603), Eagle’s minimum essential medium (MEM; Sigma-Aldrich, St. Louis, MO, USA), fetal bovine serum (FBS; Corning Cellgro, Manassas, VA, USA), 0.5% and 0.25% trypsin solution (FUJIFILM Wako Pure Chemical Corporation, Japan and Thermo Scientific, Carlsbad, CA, USA, respectively), and CELL COUNTING KIT-8 (Dojindo Laboratories, Kumamoto, Japan) were used in this study.