The Application Of Raman Spectroscopy In Methemoglobin

Hemoglobin-based oxygen carriers(HBOCs), a sort of red blood cell substitute, is the one of the current focus points in supplying enough available blood effectively. Methemoglobin concentration is one of the necessary indicators affecting the oxygen carrying capability of HBOCs. The known methemoglobin testing methods are based on spectrophotometry method. The hemoglobin modified, cross linked or enfolded with some chemical meterials will affect the testing accuracy. Raman spectroscopy is a form of vibrational spectroscopy, enabling the technique to examine hemoglobin conformation changes, with a simple, reagentless method.The solutions of different methemoglobin concentration were prepared by making hemoglobin solution reacting with different oxidants. The potassium ferricyanide was selected as a fitable kind of oxidants to establish a quantitative method to determine methemoglobin concentraion. The quantitative model is made using single linear regression method, principal component analysis method and partial least squares regression method. The coefficient correlations of these three methods are0.964,0.993and0.996, which indicate that the principal component analysis method and partial least squares regression method is better than single linear regression method. Meanwhile, Raman spectra combined with two dimensional correlation spectroscopy were used to detect the change of hemoglobin structure with different oxidative methods. The oxidation states, spin states of heme macrocyclic vibrational modes were enhanced, which indicate the porphyrin ring electron intensity and the heme core size were improved, when making hemoglobin solution reacting with the potassium ferricyanide and nitrite sodium,and making hemoglobin solution autoxidation with room temperature. The oxidation states of heme macrocyclic vibrational modes were attenuated, and spin states of heme macrocyclic vibrational modes were keep in high state, by making hemoglobin solution reacting with sodium dithionite. The oxidation state band and the spin state band were obtained to identify the difference between oxyhemoglobin, carboxyhemoglobin and methemoglobin, which suggest the potential of Raman spectroscopy as a method to study methemoglobin in different hemoglobin solutions.The results above laid a theoretical foundation and experimental basis on establish a noval quantitative method in determining methemoglobin concentraion, illustraing the molecular structural changes of hemoglobin oxidation and molecular structural differences between oxyhemoglobin, carboxyhemoglobin and methemoglobin. This study demonstrated the potential of Raman spectroscopy as a novel and convenient technique for hemoglobin measurement in red blood cell substitutes development field.