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ACS Appl Mater Interfaces. 2016 Jun 8;8(22):13830-9. doi: 10.1021/acsami.6b04658. Epub 2016 May 26.

Generation of Small Single Domain Nanobody Binders for Sensitive Detection of Testosterone by Electrochemical Impedance Spectroscopy.

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CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203, P. R. China.
Institute of Life Sciences, Southeast University , Nanjing 210018, P. R. China.
Medical School, Southeast University , Nanjing 210009, P. R. China.
Jiangsu Nanobody Engineering and Research Center , Nantong 226010, P. R. China.


A phage display library of variable domain of the heavy chain only antibody or nanobody (Nb) was constructed after immunizing a bactrian camel with testosterone. With the smaller molecular size (15 kDa), improved solubility, good stability, high affinity, specificity, and lower immunogenicity, Nbs are a promising tool in the next generation of diagnosis and medical applications. Testosterone is a reproductive hormone, playing an important role in normal cardiac function and being the highly predictive marker for many diseases. Herein, a simple and sensitive immunosensor based on electrochemical impedance spectroscopy (EIS) and Nbs was successfully developed for the determination of testosterone. We successfully isolated the antitestosterone Nbs from an immune phage display library. Moreover, one of the Nbs was biotinylated according to in vivo BirA system, which showed the highest production yield and the most stable case. Further, the EIS immunosensor was set up for testosterone detection by applying the biotinylated antitestosterone Nb. As a result, the biosensor exhibited a linear working range from 0.05 to 5 ng mL(-1) with a detection limit of 0.045 ng mL(-1). In addition, the proposed immunosensor was successfully applied in determining testosterone in serum samples. In conclusion, the proposed immunosensor revealed high specificity of testosterone detection and showed as a potential approach for sensitive and accurate diagnosis of testosterone.


electrochemical impedance spectroscopy; glassy carbon electrode; immunosensor; nanobody; testosterone

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