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A graphene oxide-based fluorescent scheme for the determination of the activity of the β-site amyloid precursor protein (BACE1) and its inhibitors

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Abstract

The enzyme cleaving the beta-site amyloid precursor protein (BACE1) is a prime therapeutic target for treatment of Alzheimer’s disease (AD). Hence, improved high-throughput methods for probing the activity of BACE1 are crucial with respect to the discovery of novel drugs for treatment and prevention of AD. We describe a graphene oxide (GO)-based fluorescent platform for the determination of BACE1 activity and screening of its inhibitors. It is found that covalent linkage of the fluorescein isothiocyanate (FITC)-labeled enzyme substrate (of sequence GTEEISEVNLDAEFRHDSGYKK) to GO results in quenching of the FITC fluorescence, but that on peptide cleavage by BACE1, FITC is released from the GO surface and fluorescence is restored. In contrast to other fluorescent assays, the method presented here shows much higher quenching efficiency and sensitivity, and it can be used at pH value of 4.5 where BACE1 exhibits higher activity. The inhibition of BACE1 activity by a potential inhibitor results in retarded fluorescence recovery. The half-maximum inhibition value of a well-known BACE1 inhibitor was found to be 82.7 nM, which is in agreement with the value reported earlier. In our perception, this method represents a valuable tool for screening of BACE1 inhibitors and discovery of AD drugs in a high-throughput screening format using microplate readers.

Covalent immobilization of a fluorescently labeled substrate for the beta-site amyloid precursor protein (BACE1) on graphene oxide (GO) induces fluorescence quenching. Peptide cleavage by BACE1 leads to the release of FITC from the GO surface; consequently, an enhanced fluorescence signal from FITC is observed

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Acknowledgments

Partial support of this work by the National Natural Science Foundation of China (Nos. 21205003, 21305004), the Joint Fund for Fostering Talents of National Natural Science Foundation of China and Henan Province (U1304205) and the Program for Science and Technology Innovation Talents at the University of Henan Province (15HASTIT001) is gratefully acknowledged.

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Authors and Affiliations

  1. College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan, 455000, People’s Republic of China

    Lin Liu, Ning Xia & Jie Yu

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  1. Lin Liu
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  2. Ning Xia
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Correspondence to Lin Liu.

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Liu, L., Xia, N. & Yu, J. A graphene oxide-based fluorescent scheme for the determination of the activity of the β-site amyloid precursor protein (BACE1) and its inhibitors. Microchim Acta 183, 265–271 (2016). https://doi.org/10.1007/s00604-015-1647-9

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  • DOI: https://doi.org/10.1007/s00604-015-1647-9

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