Colorimetric Determination of the Activity of Acetylcholinesterase and Its Inhibitors By

Electronic Supplementary Material

Colorimetric determination of the activity of acetylcholinesterase and its inhibitors by exploiting the iodide-catalyzed oxidation of 3,3’,5,5’-tetramethylbenzidine by hydrogen peroxide

Pengjuan Ni1,2, Yujing Sun1, Haichao Dai1,2, Shu Jiang1,2, Wangdong Lu1,2, Yilin Wang1,2, Zhen Li1,2, Zhuang Li 1*

1State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, People’s Republic of China

2University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China

*Corresponding author, Tel: +86 431 85262057; E-mail:

Fig. S1 The reaction between TMB, ATCI and H2O2 (1), ATCI and AChE (2), the obtained thiocholine with ox-TMB (3a) and H2O2 (3b).

Fig. S2UV-vis absorption spectra of 0.1 mM NaI in the presence of 0.5 mM TMB and 0.05 M H2O2 (1), 0.1 mM ATCh in the presence of 0.5 mM TMB and 0.05 M H2O2 (2), 0.1 mM NaI and 0.1 mM ATCh in the presence of 0.5 mM TMB and 0.05 M H2O2 (3).

Fig. S3 Effect of incubation time of ATCI with AChE on the absorbance of ATCI-TMB-H2O2 in the absence (a) and presence (b) of AChE. 10 μL of 10 mM ATCI, 10 μL of 0.5 U·mL-1 AChE; pH, 4.0; 100 μL of 5 mM TMB and 10 μL of 5 M H2O2; Reaction time, 5 min; Temperature, 37 ℃.

Fig. S4 Effect of incubation time of neostigmine with AChE on the absorbance of ATCI-TMB-H2O2 in the absence (a) and presence (b) of neostigmine. 10 μL of 10 mM ATCI, 10 μL of 0.7 U·mL-1 AChE; pH, 4.0; 100 μL of 5 mM TMB and 10 μL of 5 M H2O2; Reaction time, 5 min; Temperature, 37 ℃.

Fig. S5 Effect of pH on the absorbance of ATCI-TMB-H2O2 in the absence (a) and presence (b) of AChE. 10 μL of 10 mM ATCI, 10 μL of 0.5 U·mL-1 AChE; Incubation time, 30min; 100 μL of 5 mM TMB and 10 μL of 5 M H2O2; Reaction time, 5 min; Temperature, 37 ℃.

Fig. S6 Effect of reaction time on the absorbance of ATCI-TMB-H2O2 in the absence (a) and presence (b) of AChE. 10 μL of 10 mM ATCI, 10 μL of 0.5 U·mL-1 AChE; Incubation time, 30min; 100 μL of 5 mM TMB and 10 μL of 5 M H2O2; pH, 4.0; Temperature, 37 ℃.

Fig. S7 Effect of temperature on the absorbance of ATCI-TMB-H2O2 in the absence (a) and presence (b) of AChE. 10 μL of 10 mM ATCI, 10 μL of 0.5 U·mL-1 AChE; Incubation time, 30min; 100 μL of 5 mM TMB and 10 μL of 5 M H2O2; pH, 4.0; Reaction time, 5 min.

Fig. S8 Effect of H2O2 concentration on the absorbance of ATCI -TMB-H2O2 in the absence (a) and presence (b) of AChE. 10 μL of 10 mM ATCI, 10 μL of 0.5 U·mL-1 AChE; Incubation time, 30min; 100 μL of 5 mM TMB; pH, 4.0; Reaction time, 5 min; Temperature, 37 ℃.

Fig. S9 Effect of TMB concentration on the absorbance of ATCI -TMB-H2O2 in the absence (a) and presence (b) of AChE. 10 μL of 10 mM ATCI, 10 μL of 0.5 U·mL-1AChE; Incubation time, 30min; 10 μL of 5 M H2O2; pH, 4.0; Reaction time, 5 min; Temperature, 37 ℃.

Fig. S10 Effect of ATCI concentration on the absorbance of ATCI -TMB-H2O2 in the absence (a) and presence (b) of AChE. 10 μL of 0.5 U·mL-1 AChE; Incubation time, 30min; 100 μL of 5 mM TMB and 10 μL of 5 M H2O2; pH, 4.0; Reaction time, 5 min; Temperature, 37 ℃.

Fig. S11 The selectivity of the ATCI-TMB-H2O2 detection assay toward AChE. The concentrations of these ions and small molecules are 100 nM. The concentration of AChE is 5 mU·mL-1 (about 0.5 nM). Error bars display the standard deviations of three independent measurements.

Fig. S12 (A)Typical UV-vis absorption spectra of ATCI-H2O2-TMB solutions in the presence of 0.1 mM ATCh and different concentrations of AChE (0, 0. 5, 1.0, 2.0, 3.0, 4.0 mU·mL-1) in MDCK cell lysate solution. (B) The linear calibration plot for AChE detection in cell lysate solution.