Deciphering molecular changes (such as structure conformation) in complex systems can be challenging. If these conformation changes could be monitored in real time and modeled, it would open up new opportunities to gain a deeper understanding of signal pathways in biological systems. Through the use of confocal Raman spectroscopy, which captures the molecular fingerprint with high precision, we monitored the evolution of these changes over time. The key was to identify the spectral regions within the Raman spectrum. We employed an adaptive principal component analysis (PCA) technique to study Raman spectra and modeled strain conditions in this molecular network. Experiments were completed according to a full factorial design of experiment (DOE) approach with variable parameters including laser power density and stage temperature over the spectral range of 50-4000 /cm. Thermal effects were also introduced through the controllable micro-stage heater. We implemented this adaptive PCA technique on both individual and blended amino acids in order to highlight vibrational modes within complex samples. We examined three structurally similar branched chain amino acids to study similarities and identified specific vibrational modes that indicate molecular bending, rocking, and wagging. Results demonstrate that adaptive PCA is capable of highlighting subtle changes in molecular networks due to environmental and compositional variations. With an understanding of which data (spectral band) is more important, this speeds up computation and provides real-time analysis for monitoring conformational changes.
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.
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