Phage display library kit

Gold (III) chloride trihydrate (HAuCl₄. 3H₂O, ≥99.9), fipronil (C₁₂H₄Cl₂F₆N₄OS), (3-aminopropyl)triethoxysilane (APTES; C₉H₂₃NO₃Si, ≥98.0%), sodium chloride (NaCl), tetracycline (C₂₂H₂₄N₂O₈), glycine (C₂H₅NO₂), polyethylene glycol (PEG; C_2nH_4n+2O_n+1), sodium iodide (NaI), and bovine serum albumin (BSA) were all purchased from the renowned company Sigma-Aldrich (St. Louis, MO, USA). Trisodium citrate dihydrate (C₆H₉Na₃O₉) was provided by Kanto Chemical Co., Inc. (Tokyo, Japan). Methyl alcohol (CH₃OH, 99.5%) was purchased from Samchun Pure Chemical Co., Ltd. (Pyeongtaek-si, South Korea). LB broth was purchased from BD DIFCO. The maleic anhydride-activated plate (8-well strip) and phosphate-buffered saline (PBS; C_l2H₃K₂Na₃O₈P₂) were provided by Thermo Fisher (Waltham, MA, USA). The phage display library kit and Escherichia coli ER2738 strain were procured from New England Biolabs (Ipswich, MA, USA). The glass substrate was purchased from the Korea Testing & Research Institute (Gwacheon, Korea). The absorbance spectra were recorded on a V-770 UV–Visible/NIR Spectrophotometer (South Korea). The characterizations of the synthesized gold nanoparticles (AuNPs) were analyzed using field-emission scanning electron microscopy (FE–SEM; JITACHI S-4300 system) and field-emission transmission electron microscopy (FE-TEM; JEM2100F, JEOL Ltd., Peabody, MA, USA).

Biopanning was carried out according to the procedures of a phage display library kit (New England Biolabs Inc., Cambridge, MA), which is based on a combinatorial library of random peptide 12-mers fused to a minor coat protein (pIII) of the filamentous coliphage M13. The phage library (5.0 × 30 × 10¹⁰ transducing units (TU)) was added to the S-adenosyl-L-methionine (SAM) substrate. After incubation for 1 h at RT, the SAM substrate was washed three times with Tris-buffered saline containing 0.1% Tween 20 (TBS-T). The phages bound to Stx2B were eluted with 0.1 N Glycine-HCl buffer (pH 2.2). The eluate was neutralized with 1 M Tris–HCl buffer. The phage clones obtained were amplified through infection into host bacterial cells (E. coli ER2738). Thus, five rounds of screening were performed to enrich the targets of Stx2B. Peptide-displaying phage clones were tested by ELISA. The clones binding to Stx2B with relative higher affinity were selected from the fifth round of screening and then sequenced to identify their amino acid sequences.