Electrochemical Strategy for Sensing Protein Phosphorylation

Abstract

We herein report a novel electrochemical method in this paper to monitor protein phosphorylation and to assay protein kinase activity based on Zr(4+) mediated signal transition and rolling circle amplification (RCA). First, substrate peptide immobilized on a gold electrode can be phosphorylated by protein kinase A. Then, Zr(4+) links phosphorylated peptide and DNA primer probe by interacting with the phosphate groups. After the introduction of the padlock probe and phi29 DNA polymerase, RCA is achieved on the surface of the electrode. As the RCA product, a very long DNA strand, may absorb a large number of electrochemical speices, [Ru(NH(3))(6)](3+), via the electrostatic interaction, localizing them onto the electrode surface, initiated by protein kinase A, a sensitive electrochemical method to assay the enzyme activity is proposed. The detection limit of the method is as low as 0.5 unit/mL, which might promise this method as a good candidate for monitoring phosphorylation in the future.

Gold nanoparticles-based electrochemical method for the detection of protein kinase with a peptide-like inhibitor as the bioreceptor

“…[55][56][57][58][59] Usually, the resulting phosphorylated products could be recognized by the elements such as antiphosphorylated peptide antibodies, 19 metal ions, 20 metal complexes, [21][22][23] and nanoparticles. [24][25][26] Moreover, the use of ATP analogs as the cosubstrates (eg, ferrocene [Fc]-ATP, biotin-ATP, and adenosine 5′-[γ-thio] triphosphate [ATP-S]) can also facilitate the development of various novel electrochemical kinase biosensors. [27][28][29][30][31][32] However, most of these methods require catalyzed phosphorylation reaction to a specific peptide substrate.…”

Electrochemical detection of protein kinase activity based on carboxypeptidase Y digestion triggered signal amplification

“…The corresponding linear regressions could be expressed as I ¼-20.32 log c þ120.81 (R ¼0.9932). The detection limit was estimated to be 0.047 unit/mL (S/N ¼ 3), which was lower than those of 10 , 0.5 (Miao et al, 2012;Tan et al, 2013), 0.47 (Xu et al, 2011), 0.2 (Ji et al, 2009), 0.15 (Xu et al, 2009), 0.134 (Zhou et al, 2013), 0.1 , 0.07 and 0.061 unit/mL (Xu et al, 2012).…”

Sensitive detection of a serum biomarker based on peptide nucleic acid-coupled dual cycling reactions

“…The concentration of the ZrOCl 2 solution was adopted based on our previous work [22] as well as optimized in Fig. S2.…”