DNA-hosted copper nanoclusters/graphene oxide based fluorescent biosensor for protein kinase activity detection

Area of Science:

• Biochemistry
• Nanotechnology
• Analytical Chemistry

Background:

• Protein kinase activity (PKA) is crucial in cellular signaling pathways.
• Accurate detection of PKA is vital for understanding diseases and developing therapeutics.
• Existing PKA detection methods may lack sensitivity or require complex procedures.

Purpose of the Study:

• To develop a novel fluorescent biosensor for sensitive and specific detection of PKA activity.
• To utilize the unique properties of DNA-hosted copper nanoclusters (dsDNA-CuNCs) and graphene oxide (GO) for biosensing.
• To validate the biosensor's performance in detecting PKA and its inhibition in complex biological matrices.

Main Methods:

• Design of a dsDNA-CuNC system functionalized with an ATP aptamer.
• Utilizing graphene oxide (GO) for fluorescence quenching via Förster Resonance Energy Transfer (FRET).
• Exploiting the binding affinity changes of the ATP aptamer to GO upon ATP or PKA interaction.

Main Results:

• The biosensor demonstrated efficient fluorescence quenching by GO, which was recovered in the presence of ATP.
• PKA activity led to ATP conversion to ADP, causing reduced binding to the aptamer and subsequent fluorescence quenching.
• The sensor achieved sensitive PKA detection (0.1–5.0 U mL⁻¹) with a low limit of detection (0.039 U mL⁻¹).
• The biosensor successfully detected PKA activity in cell lysates and evaluated the inhibitory effect of H-89.

Conclusions:

• A novel, sensitive, and specific fluorescent biosensor for PKA activity detection was successfully developed.
• The integration of dsDNA-CuNCs and GO offers a robust platform for enzyme activity monitoring.
• The biosensor shows potential for applications in biochemical research and drug screening.