Reusable amperometric biosensor for measuring protein tyrosine kinase activity.
This work presents a simple, low-cost and reusable label-free method for detecting protein tyrosine kinase activity using a tyrosinase-based amperometric biosensor (tyrosine kinase biosensor). This method is based on the observation that phosphorylation can block the tyrosinase-catalyzed oxidation of tyrosine or tyrosyl residue in peptides. Therefore, the activity of p60c-src protein tyrosine kinase (Src) on the developed tyrosine kinase biosensor could be quickly determined when its specific peptide substrate, p60c-src substrate I, was used. The tyrosine kinase biosensor was highly sensitive to the activity of Src with a linear dynamic range of 1.9-237.6 U/mL and the lowest detection limit of 0.23 U/mL. Interestingly, the tyrosine kinase activity can be measured using the developed tyrosine kinase biosensor repetitively without regeneration. The inhibitory effect of various kinase inhibitors on the Src activity could be determined on the tyrosine kinase biosensor. Src-specific inhibitors, PP2 and Src inhibitor I, effectively suppressed Src activity, whereas PD153035, an inhibitor of the epidermal growth factor receptor, was ineffective. Staurosporine, a universal kinase inhibitor, inhibited Src activity in an ATP concentration-dependent manner. These results suggests that the activities of tyrosine kinases and their behaviors toward various reagents can be effectively measured using the developed tyrosine kinase biosensor.
Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, ROC.
Chung-Liang
CL
Wang
Lan-Yi
LY
Wei
Chiun-Jye
CJ
Yuan
Kuo Chu
KC
Hwang
D000255:Adenosine Triphosphate / Q000378:metabolism; D015374:Biosensing Techniques; D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D014442:Monophenol Monooxygenase / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D014443:Tyrosine; D000255:Adenosine Triphosphate; D014442:Monophenol Monooxygenase; D016392:Proto-Oncogene Proteins pp60(c-src)