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Fluorometric determination of the CCAAT/enhancer binding protein alpha by using gold nanoparticles and a labeled protein-binding DNA

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Abstract

A method is described for the determination of the CCAAT/enhancer binding protein alpha (C/EBPα) which is a regulator in adipocyte differentiation. The method is based on quenching of the red fluorescence (with excitation/emission maxima at 548/562 nm) of Cy3-labeled DNA if it becomes adsorbed on positively charged gold nanoparticles (AuNPs). Fluorescently labeled dsDNA that can bind C/EBPα is introduced as a fluorescent probes. The dsDNA is electrostatically adsorbed on the positively charged AuNPs to quench their fluorescence. In the presence of C/EBPα, it will bind dsDNA which then diffuses away. The fluorescence of the AuNPs becomes restored. The fluorescent signal increases linearly in the 0.05 to 600 ng·mL−1 μM C/EBPα concentration range, and the detection limit is 29 pg·mL−1. The method is specific and was applied to analyze cell lysates and in-situ.

Schematic representation of a fluorometric method for determination of the CCAAT/enhancer binding protein alpha (C/EBPα). Fluorescently labeled dsDNA that can bind C/EBPα is introduced as a fluorescent probes. The dsDNA is electrostatically adsorbed on the positively charged AuNPs to quench their fluorescence. In the presence of C/EBPα, it will bind dsDNA which then diffuses away. The fluorescence of the AuNPs becomes restored.

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Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (81770866, 81670773, 81770837, 81600687), Jiangsu Provincial Medical Innovation Team (CXTDA2017001), Jiangsu Provincial Medical Youth Talent (QNRC2016108), 333 high level talents training project of Jiangsu Province, Jiangsu Province “six talent peak” personal training project (YY-081) and Nanjing Technological Development Program(201715054).

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  1. Jiehua Ma and Jinlong Li contributed equally to this work.

Authors and Affiliations

  1. Nanjing Maternity and Child Health Care Institute, Women’s Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, 210004, People’s Republic of China

    Jiehua Ma, Xianwei Cui, Lianghui You, Yun Li, Juan Wen, Chenbo Ji & Xirong Guo

  2. Department of Laboratory Medicine, The Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, 210003, People’s Republic of China

    Jinlong Li

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  1. Jiehua Ma
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Ma, J., Li, J., Cui, X. et al. Fluorometric determination of the CCAAT/enhancer binding protein alpha by using gold nanoparticles and a labeled protein-binding DNA. Microchim Acta 187, 22 (2020). https://doi.org/10.1007/s00604-019-4025-1

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