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Reduced graphene oxide quenched peptide probe for caspase-8 activity detection and cellular imaging

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Abstract

Cysteinyl aspartate-specific protease 8 (caspase-8) plays a key role in various biological processes by regulating apoptosis. Therefore, this makes accurate detection and intracellular imaging of caspase-8 of great importance for drug screening, disease diagnosis, and prognostication. Here, by designing a reduced graphene oxide (rGO) quenched peptide probe, we constructed a new biosensing system for monitoring in vitro and intracellular caspase-8 activity. In this system, a fluorophore-labeled peptide and rGO were used as the substrate of caspase-8 and the fluorophore quencher, respectively. The hydrolysis of caspase-8 on the polypeptide probe substrate can generate two fragments with different lengths. The release of the short fragment labeled with the fluorophore causes recovery of the fluorescence signal (Ex/Em = 520/576 nm). Under the optimized conditions, the proposed fluorescence method exhibited a linear response range of 0.2 to 5 U·mL−1 for caspase-8 with a limit of detection (LOD) of 0.2 U·mL−1 in vitro. Furthermore, this method has been successfully applied to monitoring the upregulation of intracellular caspase-8 activity caused by tert-butyl hydroperoxide (TBHP) and fluorouracil. Flow cytometry assay indicated the positive relation between the upregulation of intracellular caspase-8 activity and cell apoptosis rate. In summary, the above results demonstrated the practical application of this method for apoptosis-related cell imaging.

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Funding

This work was partially supported by the Natural Science Foundation of Hunan Province (2021JJ30164), the National Standardization Project of Traditional Chinese Medicine (ZYBZH-Y-HUN-23), Key Research and Development Program of Hainan Province (ZDYF2022SHFS075), Science and Technology Innovation Leading Plan for High-tech Industry of Hunan Province (2020SK2043).

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Author notes

  1. The authors Xueqing Tao and Wei Zou contributed equally and were co-first authors.

Authors and Affiliations

  1. College of Biology, Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation, Hunan University, Changsha, 410082, China

    Xueqing Tao, Yan Qin, Chunyi Tong & Bin Liu

  2. Key Laboratory of Hunan Provincial TCM Administration for TCM in Obstetrics & Gynecology, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, 410008, China

    Wei Zou

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  1. Xueqing Tao
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  2. Wei Zou
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  3. Yan Qin
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  5. Bin Liu
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Correspondence to Chunyi Tong or Bin Liu.

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Tao, X., Zou, W., Qin, Y. et al. Reduced graphene oxide quenched peptide probe for caspase-8 activity detection and cellular imaging. Microchim Acta 189, 463 (2022). https://doi.org/10.1007/s00604-022-05567-0

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  • DOI: https://doi.org/10.1007/s00604-022-05567-0

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