Protein phosphorylation reagent concentration curves via Raman Spectroscopy for real-time reaction monitoring in a microfluidic reactor

Abigail H. M. Casey; Nyia E. Chusan; Caroline A. Campbell; Gregory E. Triplett

doi:10.1117/12.2531030

9 September 2019 Protein phosphorylation reagent concentration curves via Raman Spectroscopy for real-time reaction monitoring in a microfluidic reactor

Abigail H. M. Casey, Nyia E. Chusan, Caroline A. Campbell, Gregory E. Triplett

Author Affiliations +

Proceedings Volume 11087, Biosensing and Nanomedicine XII; 1108712 (2019) https://doi.org/10.1117/12.2531030
Event: SPIE Nanoscience + Engineering, 2019, San Diego, California, United States

Abstract

Protein phosphorylation is one of the most prevalent signal transduction mechanisms that occurs within cells. This biochemical reaction follows an enzymatic reaction mechanism where the enzyme or kinase facilitates the transfer of the phosphate group from adenosine triphosphate (ATP) to the substrate protein. By monitoring this reaction in real-time, outside of the cell, the knowledge gained can be applied towards intracellular research in the future. Our goal is to combine microfluidic reactor technology with confocal Raman spectroscopy to investigate biochemical reactions such as protein phosphorylation in order to profile the reaction along the reactor path. By developing an approach that can monitor structural and conformational changes of proteins during biochemical reactions we can provide insight towards signal transduction mechanisms. Our reactor design is based off fluid dynamic principles and continuous reactor design equations. The change in concentration of a reagent during a reaction can be determined by a change in the intensity of its spectral response. The individual reagents for this particular protein phosphorylation reaction include protein kinase A, casein, ATP, a pseudosubstrate, as well as the three phosphorylatable amino acids: L-serine, L-threonine and L-tyrosine. Raman spectroscopy of varying concentrations of each individual reagent will quantify a change in concentration during the reaction. Concentration calibration curves were acquired on solutions inside the microreactor. Lower limit concentration detectability of the Raman instrument was also determined. Full Raman characterization of solid individual reagents was employed as a baseline for comparison of concentration measurements to monitor changes in reagents.

Citation Download Citation

Abigail H. M. Casey, Nyia E. Chusan, Caroline A. Campbell, and Gregory E. Triplett "Protein phosphorylation reagent concentration curves via Raman Spectroscopy for real-time reaction monitoring in a microfluidic reactor", Proc. SPIE 11087, Biosensing and Nanomedicine XII, 1108712 (9 September 2019); https://doi.org/10.1117/12.2531030

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