Electrogenerated Chemiluminescence Bioassay of Two Protein Kinases Incorporating Peptide Phosphorylation and Versatile Probe

Abstract

A sensitive electrogenerated chemiluminescence (ECL) bioassay was developed for the detection of two protein kinases incorporating the peptide phosphorylation and a versatile ECL probe. Cyclic adenosine monophosphate-dependent protein kinase (PKA) and casein kinase II (CK2) were used as proof-of-concept targets while a PKA-specific peptide (CLRRASLG) and a CK2-specific peptide (CRRRADDSDDDDD) were used as the recognition substrates. Taking advantage of the ability of protein A binding with the Fc region of a variety of antibodies with high affinity, a ruthenium derivative-labeled protein A was utilized as a versatile ECL probe for bioassay of multiple protein kinases. A specific peptide substrate toward target protein kinase was first self-assembled on the surface of gold electrode and then serine in the specific peptide on the electrode was phosphorylated by target protein kinase in the presence of adenosine-5′-triphosphate. After recognition of the phosphorylated peptide by monoclonal antiphosphoserine antibody, the versatile ECL probe was specifically bound to the antiphosphoserine antibody on the electrode surface. The ECL bioassay was developed successfully in the individual detection of PKA and CK2 with detection limit of 0.005 U/mL and 0.004 U/mL, respectively. In addition, the ECL bioassay was applied to quantitative analysis of the kinase inhibitors and monitoring drug-triggered kinase activation in cell lysates. Moreover, an ECL imaging bioassay using electron-multiplying charged coupled device as detector on the gold electrode array was developed for the simultaneous detection of PKA and CK2 activity from 0.01 U/mL to 0.4 U/mL, respectively, at one time. This work demonstrates that the ingenious design and use of a versatile ECL probe are promising to simultaneous detection of multiple protein kinases and screening of kinase inhibitor.

Confined Electrochemiluminescence Generation at Ultra-High-Density Gold Microwell Electrodes

“…Thus, the signals generated by individual electrodes in the whole array can be all read at once and spatially resolved, allowing the simultaneous detection of multiple targets in a single workflow (Deiss et al, 2009;Zhang et al, 2014;Cui et al, 2020). Previous studies have mainly focused on the application of microelectrode arrays for the quantitative determination of biomolecules, such as glucose, nucleic acid, proteins and cells, suggesting the porous electrode array is very useful in biosensing (Deiss et al, 2009;Liu et al, 2016;Xu et al, 2016;Xia et al, 2018). While those concerning the ECL reaction mechanisms are scarce (Chovin et al, 2004).…”

Integrating Highly Efficient Recognition and Signal Transition of g-C3N4 Embellished Ti3C2 MXene Hybrid Nanosheets for Electrogenerated Chemiluminescence Analysis of Protein Kinase Activity

“…Recently, an ECL-based immunosensor for the kinase activity has been reported with wide linear range . In addition, the ECL biosensor for multiple kinase activity detection was also developed using a ruthenium-labeled protein A as an ECL probe which could bind to the Fc region of a variety of antibodies with high affinity . However, most of these methods involve the employment of biorecognition units such as antibodies and protein A and so on, which are limited in the storage stability, sensitivity, and the false positive analysis from the cross-link immunoreactions, and it is difficult to explore for the analysis of uncertain kinase.…”

Recent advances in electrogenerated chemiluminescence biosensing methods for pharmaceuticals

“…The relative ECL intensity from the first wave is significant, especially in diluted Ru(bpy) 3 2+ solutions (less than approximately micromolar) containing ∼0.1 M TPA. Thus, for the low concentrations of analytes such as in immunoassays and detection of drugs and biomakers with Ru(bpy) 3 2+ as an ECL label, the bulk of the ECL signal obtained in this system probably originates from the first ECL wave at Au electrode or from the second ECL wave at carbon electrode [27] , [28] , [29] , [30] , [31] , [32] . In recent years, cyclometalated iridium (III)-complexes have received much attention, since these complexes have high ECL efficiencies and low emission potentials compared with Ru(II) complexes [33] , [34] .…”