N γ maleimidobutyryl oxysuccinimide ester gmbs

TNT solution dissolved in pure DMF (N,N-dimethylformamide) was obtained from Chugoku Kayaku, Co., Ltd., Kure, Japan. The concentration of the received TNT solution was 10.3 mg/mL and was freshly diluted as required with phosphate-buffered saline (PBS, 0.1 M, pH 7.4) containing 0.05% Tween 20 (T) and 5% DMF before each measurement. Five kinds of TNT analogues, research and development explosive (RDX) solution dissolved in pure DMF (10.3 mg/mL) were purchased from Chugoku Kayaku, Co., Ltd., Kure, Japan. 2,4-Dinitrophenyl glycine (DNP-glycine), 2,4-DNT, 2,6-DNT, and 4-nitrobenzoyl-glycyl-glycine were purchased from Tokyo Chemical Industry, Tokyo, Japan. 3-Aminopropyltriethoxysilane (APTES) 99% was purchased from Sigma-Aldrich, St. Louis, MO, USA. N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS) was purchased from Thermo Fisher Scientific, Rockford, IL, USA. L-cysteine was purchased from Sigma-Aldrich. The SPR Au chip was purchased from Biacore, GE, Uppsala, Sweden. Sodium hydroxide solution (NaOH) and potassium hydroxide solution (KOH) were purchased from Wako Pure Chemical Industries, Tokyo, Japan. All other chemicals were purchased either from Tokyo Chemical Industry, Tokyo, Japan or Wako Pure Chemical Industries, Tokyo, Japan. All aqueous solutions were prepared by MilliQ deionized water (18 MΩ) from MilliQ-system (Millipore Corporation, Billerica, MA, USA).

The paper-based immunoaffinity devices were produced by immobilizing anti-CD63 antibodies on grade 1 Whatman filter papers (Supplementary Figure S2). Initially, the targeted reaction zone of this substrate was treated with 50 μL of 4% (v/v) 3-mercaptopropyl tri-methoxysilane (Sigma-Aldrich, United States ) in 99% ethanol (ECHO chemical, Taiwan) and incubated for 30 min. Subsequently, the reaction zone was treated with 50 μL of N-γ-maleimidobutyryloxy succin-imide ester (GMBS) (Thermo Scientific, United States ) (0.01 μmol in 99% ethanol) and incubated for 15 min. The device was then incubated in 10 μg/ml Neu-trAvidin (Thermo Scientific, United States ) solution in PBS (Thermo Scientific, United States ) at 4°C for 1 h. Blocking was conducted with 1% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich, United States ) in phosphate-buffered saline (PBS) for 10 min and repeated 3 times. Finally, the reaction zone was introduced with 20 μL of biotinylated mouse anti-human anti-CD63 antibody (BioLegend, United States ) and incubated for 10 min 3 times.