Aptamer based fluorescence biosensor for protein kinase activity detection and inhibitor screening

- We firstly used Cu NCs as a label-free and sensitive fluorescent method for PKA activity.
- The CuNCs was formed by ATP aptamer which can combine with ATP.
- A good linear response for PKA was found with a much lower detection limit.
- This probe was further used for PKA inhibitor detection.
- It was applied to the investigation of drug-induced PKA activation in HeLa cells.

Herein, we firstly used double-strand-templated DNA Cu nanoclusters (Cu NCs) as a simple, lable-free and sensitive fluorescence (FL) probe to detect protein kinase (PKA) activity. This method was based on the strong interaction between adenosine triphosphate (ATP) aptamer and ATP. When ATP was added, fluorescent Cu NCs could not be formed due to the lack of effective substrate and the fluorescence of Cu NCs decreased. However, when PKA was added, the fluorescence of Cu NCs recovered because that ATP had been translated into ADP by PKA and ADP can not combine with ATP aptamer. We can effective monitored the activity of protein kinase according to the variation of fluorescence signal in the range of 0.1-1000 mU/μL. The detection limit (LOD) is 0.041 mU/μL. We also used this method to detect protein kinase inhibitor H-89. In addition, this method was also used to explore the activity of drug-induced PKA in Hela cells, which makes the sensor had a great application value in biochemistry and targeted kinase drug discovery research.

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