Electrochemical Monitoring of an Important Biomarker and Target Protein: VEGFR2 in Cell

Supplementary Information

Electrochemical Monitoring of an Important Biomarker and Target Protein: VEGFR2 in Cell Lysates

Tianxiang Wei, Wenwen Tu, Bo Zhao, Yaqian Lan, Jianchun Bao, and Zhihui Dai*

Jiangsu Collaborative Innovation Center of Biomedical Functional Materials and Jiangsu Key Laboratory of Biofunctional Materials, School of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, P. R. China

*Corresponding author. Fax: +86-25-85891051; Tel: +86-25-85891051; E-mail:
ELISA assay procedure for quantification of VEGFR-2 protein: A double sandwich ELISA (Duo Set ELISA, R&D Systems) was used to measure the concentration of VEGFR2 following the manufacturer instructions with minor changes. Briefly, 96-well microplates were coated with the capture antibody and incubated overnight at room temperature. Coating was followed by blocking non-specific binding sites of the plates by 3% BSA in PBS at room temperature for 1 h. After that, samples and standards were incubated for 2 h at room temperature, followed by the addition of the biotinylated detection antibody and incubated for 2 h at room temperature. Next, a solution of Streptavidin-HRP was added to each well and incubated for 30 minutes at room temperature. After washing, TMB HRP-substrate solution was added and incubated for 20 minutes followed by the addition of a stop reagent (0.12 M hydrochloric acid). A microplate reader set to 450 nm and a reference at 540 nm was used to determine the optical density of each well immediately. All samples were assayed in duplicate.

Optimization of detection conditions: A variety of factors would affect the electrochemical response of the biosensor. Herein, we explored three factors, including the density of chitosan-RGO coated on the surface of electrode (Figure S3A), the incubation time of VEGFR2 (Figure S3B) and the concentration of H2O2 in the detection solution (Figure S3C). As mentioned in Figure 3, the chitosan-RGO composite coated on the electrode surface accelerated electron transfer. But, the amount of the chitosan-RGO had a great influence on electrochemical responses. In the case of adding a certain volume of chitosan-RGO suspension, as shown in Figure S3A, with increasing the density of chitosan-RGO from 0.15 to 1.0 mg mL−1, the peak current increased to a maximum at 0.25 mg mL−1 chitosan-RGO, which might own to the broadening of the conductive electrode area. And then the peak current tended to decrease obviously from 0.25 to 1.0 mg mL−1, which was probably attributed to the limited electron transfer inside a thicker film. Hence, a density of 0.25 mg mL−1 chitosan-RGO was chosen to modify the electrode. The incubation time for capturing VEGFR2 antigen was another important parameter. Figure S3B showed that the peak current increased with increasing incubation time of VEGFR2 antigen and tended to level off after 60 min, indicating the saturated formation of the sandwich immunocomplex on the electrode surface. Therefore, 60 min was selected as the incubation time of VEGFR2. The electrochemical response was also related to the concentration of H2O2. As revealed in Figure S3C, the current of the biosensor reached a maximum value at 4 mM H2O2. Hence, the optimal concentration of H2O2 was 4 mM.

Figure S1. A) UV-vis absorption spectra and B) Raman spectra of dispersions of a) chitosan-GO and b) chitosan-RGO.

Figure S2. A) CVs of Ab2/Ag/BSA/Ab1/chitosan-RGO/thionine/GCE in 0.01 M pH 7.4 PBS at 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mV s−1 (from lowest to highest peak currents). B) Plot of peak current vs. scan rate.

Figure S3. Effects of A) the density of chitosan-RGO coated on the surface of electrode B) incubation time of VEGFR2 and C) the concentration of H2O2 in the detection solution on the DPV responses of the immunosensor toward 43 pM VEGFR2. The error bars were calculated from three times parallel experiments.

Figure S4. The calibration curve obtained by using the ELISA kit for detection of VEGFR2 from 0.4 pM to 52 pM. OD = 0.1660 + 0.0208 [VEGFR2] (pM) in the linear range from 0.9 pM to 43 pM with a correlation coefficient of 0.99.

Figure S5. The stereo views of the docking pose of VEGFR2 with A) Sorafenib and B) Vandetanib.

S5