Supporting information

Nanobody-based electrochemical immunoassay for Bacillus thuringiensis Cry1Ab toxin by detecting the enzymatic formation of polyaniline

Min Zhu a, b, 1, Min Li a, 1, Guanghui Li a, b, Zikai Zhou a, Hong Liu e, Hongtao Lei f, Yanfei Shen d, *, Yakun Wan a, b, c *

a Institute of Life Sciences, Southeast University, Nanjing 210096, PR China

b CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences

c Jiangsu Nanobody Engineering and Research Center, Nantong 226010, PR China.

d Medical School, Southeast University, Nanjing 210009, PR China

e Plexera LLC, WA 98072, USA

f GuangdongProvincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou510642,PR China

* Corresponding authors: Yanfei Shen PhD, Professor, Dingjiaqiao NO.87, Southeast University, Nanjing 210009, PR China, Email: (Y. Shen)

Yakun Wan PhD, Professor, 555 Zuchongzhi Road, CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, PR China, Tel.: +86 21 50805906; Email: (Y. Wan)

1 These authors have equally contributed to this work.

Figure S1. The whole procedure. Schematic representation of the strategy to select the Cry1Ab-specific VHHs and to apply the detection approach. VHH genes from an immunized dromedary were sub-cloned into pMECS phagemid and transformed into TG1 cells to construct a VHH library. Cry1Ab-specific VHHs were selected form the library after several round of bio-panning by phage display technology. Nanobodies identified to recognize different epitopes were used to electrochemical detection after coupled with biotin or HRP.

Figure S2. VHHs specific to Cry1Ab toxin were selected from a phage displayed nanobody Library. (A) SDS-PAGE for Cry1Ab toxin protein. (B) 24 colonies were randomly selected to estimate the correct insertion rate by performing PCR. (C and D) Periplasmic extract ELISA for 95 clones randomly selected from round one and round two bio-panning and 12 clones from the second round were identified as the positive clones. The ratio higher than 2 was considered as positive.

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