1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride

To investigate interactions between NT5B and CT5B proteins, a Biacore T200 biosensor with Biacore Control software was used (GE Healthcare). Initially 25 μg/ml of CT5B diluted in 50 mm sodium acetate, pH 5.5, was immobilized onto a CM5 sensor chip by amine coupling using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride, N-hydroxysuccinimide, and ethanolamine-HCl (GE Healthcare). The immobilized CT5B gave ∼3400 response units. To perform a pH scan experiment, purified NT5B protein (50 nm) was injected over the immobilized CT5B in varying pH buffers containing 5 mm CaCl₂ and 0.05% Tween 20 for 120 s at a flow rate of 30 μl/min and dissociation time of 60 s. The CM5 sensor chip was regenerated after each sample application with 10 mm EDTA, pH 7.4, at a flow rate of 30 μl/min for 30 s and stabilization of 1 min after regeneration. For single cycle kinetic analysis, 0, 5, 10, 15, 20, and 40 nm NT5B analyte was flowed over the immobilized CT5B in HBS containing 5 mm CaCl₂ and 0.05% Tween 20, at pH 6 or 7.4. Curves were fitted using 1:1 Langmuir association/dissociation model (Biacore Evaluation 4.1 software; GE Healthcare) to obtain kinetic values of binding affinity.

Tris(2-pyridylmethyl)amine (TPMA, Sigma Aldrich, MO, USA), copper chloride (CuCl₂, Nacalai Tesque, Inc., Japan), and ascorbic acid (Wako Pure Chemical Industries, Japan) were used in the ATRP reaction. MES and HEMA were obtained as monomers from Sigma Aldrich (MO, USA). Bis[2-(2′-bromoisobutyryloxy)undecyl]disulfide (DTBU) from Sigma Aldrich (MO, USA) was prepared to form an SAM as an initiator for ATRP. N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3′-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased as an amine coupling kit from GE Healthcare Bio-sciences (Uppsala, Sweden). 2,4-Dinitrophenyl-e-aminocaproyl-NHNH2 (DNP-Hdrz) was prepared as a TNT analog from Biosearch Technologies, Inc. (USA). Mouse anti-TNT monoclonal antibody (anti-TNT antibody) was obtained from Strategic Biosolutions (DE, USA). A TNT aqueous solution with a concentration of 21.8 ppm was purchased from Chugoku Kayaku (Hiroshima, Japan). Bovine serum albumin (BSA, Wako Pure Chemical Industry, Japan) and lysozyme (Sigma Aldrich, MO, USA) were used to evaluate non-specific adsorption. All other chemicals were purchased either from Tokyo Chemical Industry (Japan), Wako Pure Chemical Industries, Inc. (Japan), or Kanto Chemical, Co. (Japan). All aqueous solutions were prepared with Milli-Q water obtained from a Milli-Q system (Millipore, MA, USA).