The Chemistry Of NHS With O-Acylisourea

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Because of the chemical inertness of MWCNTs, it is essential to create reactive groups on the surface for further modification [15]. The carboylic groups on the MWCNTs provide ideal anchoring point for the covalent immobilization of enzyme using EDC/NHS [15]. It has been demonstrated that sonicating MWCNTs in a mixture of acid solution opens the tube caps and forms carboxylic groups at the defect sites along the side walls [36].
Fig. 1 shows the FTIR spectra of MWCNTs and acid-treated MWCNTs. The appearance of sharp peaks at 1642 cm-1 indicates the presence of carbonyl groups (C=O) of the quinine type units along the side walls of the nanotubes [37], and its intensity decreased after acidification. Two noticeable peaks appeared at 1737 and
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In general, one of the EDC double bonds reacts with a carboxyl group to form an amine-reactive O-acylisourea intermediate, which is prone to hydrolysis to regenerate the acid group, and the rearrangement of O-acylisourea forms unreactive N-acylisourea. The reaction of NHS with O-acylisourea results in the formation of NHS-ester, which is more reactive towards primary amines and more resistant to hydrolysis, and it also undergoes minimal formation of N-acylurea [40]. Therefore, NHS is usually used with EDC to increase the efficiency of EDC-mediated enzyme immobilization. The formation of NHS-ester is critically affected by pH; the carboxyl groups have poor reactivity to EDC/NHS at pH <4.5, and NHS-esters are more susceptible to hydrolysis at alkaline pH values [18,19,20,41]. The amidation reaction of NHS-ester with amino groups is known to be most efficient at the pH values between 6 and 8; amino groups exhibit poor nucleophilicity to NHS-ester at low pH because of the formation of protonated amino groups, but amino groups have strong nucleophilicity, whereas NHS esters are unstable at high pH [18,19,20,41].
In this study, a two-step immobilization procedure was used: activation of carboxylated MWCNTs (NHS-ester formation) and coupling of GOx onto MWCNTs via the NHS-ester. The carboxylated MWCNTs were reacted with EDC/NHS at pH 6.0 for 3 h. The coupling reactions were performed at two pH levels (pH 6.0 and pH 7.4) for 3
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Furthermore, it has been argued that proteins can physically adsorb onto MWCNTs in the EDC/NHS-mediated immobilization [15]. Therefore, it was necessary to investigate whether the observed GOx immobilization occurred through covalent attachment or physical adsorption. The inclusion of Tween-20 in the post-reaction washes or its addition to the reaction mixture is known to effectively remove any physically adsorbed proteins during immobilization [42]. Accordingly, the effect of Tween-20 was examined for removing physically adsorbed GOx: the carboxylated MWCNTs were activated by EDC (47 mM) and NHS (165 mM) and then reacted with GOx (3.5 mg/mL). After separation, GOx-MWCNTs were washed with buffer containing different concentrations (0–5 g/L) of Tween-20, and GOx detachment was monitored. As shown in Fig. 3, an increase in the detachment of GOx was observed from GOx-MWCNTs as the concentration of Tween-20 increased. GOx loading was only 2.7 ± 0.1 mg-GOx/mg-MWCNTs after washing with buffer containing 5 g/L of Tween-20, whereas a value of 6.4 ± 0.4 mg-GOx/mg-MWCNTs was observed after washing with buffer only. For comparison, GOx immobilization was also performed in the same conditions except that carboxylated MWCNTs were not activated by EDC/NHS. GOx loading was 2.2 ± 0.2 mg-GOx/mg-MWCNTs after washing with buffer only, and it decreased

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