Protein kinases are key players in many aspects of cell activities such as growth, differentiation, migration, and in response to external stimuli [1
]. Protein kinases also play critical roles in tumorogenesis, thus they have become important targets for drug development [4
]. Kinase activity involves structural modification of substrate molecules by covalently conjugating a phosphate group. This structural change can be detected by radioactive labeling, or by antibody recognition. Current practice to monitor cell kinase activity are often time consuming and labor intensive. Methods involving radioactive material labeling and/or antibodies usually take 1–2 days to accomplish. Therefore a simple, quick method to detect kinase activity is highly desired.
Raman spectroscopy allows direct monitoring of structural changes in macromolecules without further labeling/modification or denaturing of the target. However this method is usually not sensitive enough for biological systems. Various methods of enhancement were developed to expand the detection limit. Among these, enhancement with heavy metal colloid nanoparticles (termed surface enhanced Raman scattering, SERS) was particularly interesting. With this method, probing single molecules absorbed onto a single silver nanoparticle or a single carbon nanotube was achieved [6
]. The sensitivity of this method has been shown to be similar to that of the fluorescent detection method for the R6G molecule [6
]. In this previous work, the amplification magnitude of SERS was estimated to be as high as 1014
Through different enhancement methods, the possibility of differentiating phosphorylated and non-phosphorylated peptides was explored recently [9
]. Using synthetic, pure peptides at high concentration, characteristic Raman spectra on phospho-serine and phospho-tyrosine were reported [9
]. However, a real test on kinase activity has not been reported. One possible hurdle could be that it is technically difficult to reach an ideal platform that combines signal amplification and the biological nature of kinase reaction.
Here we extended this line of research by directly measuring kinase activity via SERS. We wish to combine the highest sensitivity reported on SERS (i.e. single molecule detection), with a flexible platform that is suitable for biological molecular reaction. To this end, we employed a silver nanoparticle approach with enzyme substrate attached to the surface, and enable enzyme reaction in the liquid/solid interface. We first demonstrated the feasibility of this method using purified kinase in an in vitro reaction system. We then showed that it is also possible to measure kinase activity in crude cell lysate. Thus a SERS-based detection method can be readily applied to measure kinase activity in real biological system.