A facile method for detecting calcium/calmodulin-dependent protein kinase using radio phosphorylation of a GBP-fused enzyme substrate in a lab-on-a-chip

A protein-fused substrate for calcium/calmodulin-dependent protein kinase (CaMKII) was constructed by genetically fusing a gold binding polypeptide (GBP) to the putative spermidine synthase (PSPD) of Selenomonas ruminantium. DNA encoding the CaMKII substrate Autocamtide-2, the amino acid sequence of which is KKALRRQETVDAL, was cloned and fused to the C-terminal 32 kDa of PSPD. In the present study, the GBP-PSPD-fusion Autocamtide-2 substrate (GBP-SP-AC2) was used to detect signal intensities reflecting CaMKII action, on gold-coated glass slides. Construction of a lab-on-a-chip (LOC) employing radioisotopes (RIs) requires the appropriate design, fabrication, and testing of plastic microfluidic devices allowing on-chip substrate preparation and employment of protein microarrays. We investigated the feasibility of RI detection techniques used to measure phosphorylation of a GBP-fused substrate, in turn facilitating highly sensitive detection of CaMKII, employing an LOC. Our LOC device designed for use in applications employing radioisotopes is architecturally simple, affords rapid testing, is cost-effective, and generates minimal amounts of radioactive waste. This strategy can be used in the general development of LOCs for high-throughput screening in biological and medical research.