Electronic Supplementary Material
Immunoassay for netrin 1 via a glassy carbon electrode modified with multi-wall carbon
nanotubes, thionine and gold nanoparticles
Wailan Xu1, Junlin He1, Liuliu Gao, Jing Zhang, Chao Yu*
Institute of Life Science and School of Public Health, Chongqing Medical University, Chongqing
400016, P. R. China
1
Junlin He and Wailan Xu contributed equally to this work.
*Corresponding author: Email: yuchaom@163.com, Phone: (86) 23-68485589, Fax: (86)
23-68486294
Optimization of method
To achieve the optimal performance immunoassay of our developed immunosensor, many
parameters had been chosen to optimize. The pH values of the substrate solution had a significant
effect on the electrochemical behavior of the immunosensor, because immune proteins are
influenced by acidity of the buffer. Highly acidic or alkaline surroundings would damage the
structure of the immobilized protein. Moreover, thionine is a pH-dependent dye, so the
electrochemical behavior of Thionine modification on GCE also can be influenced by pH values.
To sum up, the pH of solution is a crucial factor. The trend of the dependence pH of phosphate
buffer on the immunosensor response is shown in Fig. S1A. As can be seen, at the ranges from 5.0
to 8.0 of pH, the current was increased to the maximum at 7.4, whereas further increases of pH
led to the decreases of the current, which is similar to previous reported [1]. Hence, a pH of 7.4
has been selected as the working solution.
Thionine is a known redox probe that can facilitate the charge transfer between the electric
materials and the GCE surface. Furthermore, we have found that the concentration of thionine has
a great impact on both the peak shape and the peak current value. Therefore, the concentration of
thionine was investigated in the range of 0.1 to 1.0 mg mL-1. As presented in Fig. S1B, the highest
response was found at 0.25 mg mL-1, and the current then decreased at the concentration above
0.25 mg mL-1. Thus, 0.25 mg mL-1 is chosen as optimal concentration of thionine.
On the other hand, the incubation time of thionine and immunoreaction had a significant effect
on the sensitivity of our immunosensor. The incubation time of antigen and antibody is generally
depends on the kinetic characteristics of immunoreactions between antigens and antibodies. And
the incubation time of thionine will affect the adsorption quantity of thionine, further affect the
electrochemical response. In this part, the current variation was used to monitor the effect of the
incubation time on our developed immunosensor. As shown in Fig. S1C and 1D, it was found
obviously that the current variation value reached maximum at 105 min and 50 min, respectively.
Therefore, we selected 105 min and 50 min as the optimum incubation time of thionine and
immunoreactions for the immunoassay.
Fig. S1. The effect of (A) pH, (B) thionine concentration, (C) incubation time of thionine and (D)
antigen and antibody immunoreactions.
References
1. Ou C, Yuan R, Chai Y, Tang M, Chai R, He X (2007) A novel amperometric immunosensor
based
on
layer-by-layer
assembly
of
gold
nanoparticles-multi-walled
carbon
nanotubes-thionine multilayer films on polyelectrolyte surface. Anal Chim Acta 603:205-213.