Switchable "On-Off" electrochemical technique for detection of phosphorylation.
In this paper, we report a simple but ultrasensitive electrochemical method for the detection of phosphorylation and the activity of protein kinase A (PKA). In this detection system, the substrate peptides form a compact and positively charged self-assembly monolayer (SAM) on an electrode surface. It will block the positively charged electrochemical probe [Ru(NH3)5Cl]2+ from getting access to the electrode, keeping the "Off" state of the detection system. After the phosphorylation by the enzyme, the SAM becomes loose due to the generation of phosphate groups. Meanwhile, due to the electrostatic interaction between the negative phosphate groups and positively charged SAM, [Ru(NH3)5Cl]2+ molecules can move and get close to the electrode surface. As a result, electrochemical signal is observed and the system is at "On" state. Furthermore, the switch between the "On" and "Off" states can be achieved by the catalysis of PKA and alkaline phosphatase, respectively. The electrochemical response can be used to assay the PKA activity with an extraordinarily low detection limit of 1×10(-4) unit/mL and wide linear range from 1×10(-4) to 1×10(-2) unit/mL. This assay can also be performed by using serum samples with high specificity and reproducibility.
Wang
Jing
J
Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 210093 Nanjing, China.
Shen
Min
M
Cao
Ya
Y
Li
Genxi
G
D015374:Biosensing Techniques / Q000295:instrumentation*; D003217:Conductometry / Q000295:instrumentation*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis* / Q000737:chemistry*; D004789:Enzyme Activation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D010766:Phosphorylation; D012815:Signal Processing, Computer-Assisted / Q000295:instrumentation*
D017868:Cyclic AMP-Dependent Protein Kinases