Phosphorylation is the most important post-translational modification of proteins in eukaryotic cells and it is catalysed by enzymes called kinases. The balance between protein phosphorylation and dephosphorylation is critical for the regulation of physiological processes and its unbalance is the cause of several diseases. Label-free biosensing techniques can provide improved devices for high throughput drug discovery platforms. We have developed two versatile methods to detect the release of protons (H+) associated with the protein phosphorylation catalysed by kinases . The first approach is based on the pH-sensitive response of oxide semiconductor interfaces and the second method detects the pH changes in phosphorylation reaction using a commercial micro pH electrode. Detection on pH-sensitive silicon nitride (Figure 1) shows a remarkably high sensitivity and response. A good correlation was observed between our hypothesis and the experimental results. Using phosphorylation of myelin basic protein by PKC-α kinase as a case study, silicon nitride based electrolyte insulator semiconductor capacitor structures revealed a change in gate voltage of 37 mV upon phosphorylation as compared to less than 2 mV in control experiments wherein kinase activity was inhibited. The commercial micro-pH meter detected a low change, at around 1.5% of change in pH as compared to the change in pH observed on Si3N4. This is because direct detection of pH variations is challenging due to the buffering capabilities of the solution and the technological difficulties in developing stable micro-pH electrodes. Nevertheless it is still able to significantly distinguish between the phosphorylated and non-phosphorylated sample proved by statistical analysis. These techniques can be readily adopted for multiplexed arrays and high throughput analysis of kinase activity, which will represent an important innovation in biomedical research and drug discovery.
|Publication status||Published - 2014|
|Event||24th Anniversary World Congress on Biosensors - Melbourne, Australia - |
Duration: 27 May 2014 → 30 May 2014
|Conference||24th Anniversary World Congress on Biosensors - Melbourne, Australia|
|Period||27/05/14 → 30/05/14|
Bhalla, N., Di Lorenzo, M., Pula, G., & Estrela, P. (2014). Novel tools for protein phosphorylation analysis based on proton release detection. Abstract from 24th Anniversary World Congress on Biosensors - Melbourne, Australia, .