A FRET assay for the quantitation of inhibitors of exonuclease EcoRV by using parchment paper inkjet-printed with graphene oxide and FAM-labelled DNA

Abstract

A graphene oxide (GO)-based cost-effective, automatted strip test has developed for screening of inhibitors of endonuclease EcoRV. The method involves the use of GO and a DNA substrate for EcoRV that contains both an ssDNA region for binding of GO and a fluorescein amidite (FAM)-labelled dsDNA. All the components were inkjet printed on a piece of parchment paper. The ssDNA region binds to the surface of GO and anchors so that the fluorescence of FAM is quenched. The parchment paper strip is then incubated with a sample containing EcoRV which causes enzymatic hydrolysis, and dsDNA was separated from the GO. As a result, green fluorescence is generated at the reaction spot. Enzyme activity can be measured in the presence and absence of aurintricarboxy acid acting as an EcoRV inhibitor. This method excels by its need for 2–3 orders less reagents compared to the standard well plate assay. Thus, it is an efficient platform for GO-based screening of EcoRV enzyme inhibitors.

A graphene oxide (GO)-based endonuclease EcoRV inhibition FRET assay using inkjet printing was developed. Printing of GO along with assay reagents has a beneficial effect on the enzymatic reaction on paper. This method was successfully applied to evaluate EcoRV inhibitor activity.

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Acknowledgements

This work was supported by National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) (2016R1A4A1010796 and 2018M3A7B4071235). We are grateful to the Research Institute of Pharmaceutical Sciences at Seoul National University for providing experimental equipment and Brain Korea 21 plus (BK 21 plus). The authors declare no competing financial interests.

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Correspondence to Joon Myong Song.

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Lee, J., Samson, A.A.S., Yim, Y. et al. A FRET assay for the quantitation of inhibitors of exonuclease EcoRV by using parchment paper inkjet-printed with graphene oxide and FAM-labelled DNA. Microchim Acta 186, 211 (2019). https://doi.org/10.1007/s00604-019-3317-9

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Keywords

  • Single-shot assay
  • Drug screening
  • Inkjet printing
  • Quantitative analysis
  • Paper strip