基于拉曼光谱方法研究不同剪切力下的红细胞亚损伤

利用共聚焦拉曼光谱技术，对人工心脏泵不同剪切应力下受到亚损伤的红细胞进行实验研究，验证拉曼光谱对红细胞亚损伤程度的评估能力，为血液损伤评价提供了一种新的思路。实验采集血红蛋白和红细胞的拉曼光谱标准谱图并进行对比分析，以确定红细胞谱图特征峰的归属。用血液剪切力试验平台对测试血样施加暴露时间为1 s，大小分别为0，50，100，150，200，250和300 pa的剪切力。利用共聚焦拉曼仪器，在10倍长焦物镜，532 nm激光光源波长，积分时间10 s，积分次数2次，2.5 mW功率下采集剪切应力作用后的红细胞拉曼谱图。通过归一化的方法对比红细胞的拉曼谱图变化，评估红细胞亚损伤的程度，运用曲线拟合方法对特征峰和剪切应力进行拟合，验证拉曼光谱对红细胞亚损伤的评估能力。对比血红蛋白和红细胞的拉曼光谱标准谱图发现，红细胞谱图能够反映血红蛋白的内部结构。且结果表明，拉曼光谱法可以用于区分不同剪切应力下亚损伤的红细胞，推断剪切应力可以透过细胞膜从而影响到其内部的血红蛋白结构。且随着剪切力的增大，1 376 cm-1位置左侧谱线呈现明显抬高趋势，1 549和1 604 cm-1位置的峰强增高，1 639 cm-1位置的氧浓度标记带ν₁₀振动谱带减弱。其中1 549 cm-1位置的峰强为亚铁离子高自旋带，在不同剪切力的作用下，峰强差异表现最明显，与剪切应力呈明显的正向线性关系，拟合效果良好。拉曼光谱法检测样本处理简单、耗时短、操作简便、重现性好，且可以精确的检测到细胞内部结构的细微变化，可以评估红细胞的亚损伤程度，弥补了传统评价溶血的方法的不足，为人工心脏泵血液损伤评价提供了新的技术手段，拓宽了拉曼检测方法的应用领域。

Study on Erythrocyte Sublethal Damage Under Different Shear Stress Based on Raman Spectroscopy

In this study, confocal Raman spectroscopy was used to study the Sublethal damage erythrocytes of artificial heart pump under different shear stress to verify the ability of Raman spectroscopy to evaluate the degree of erythrocyte Sublethal damage. It provides a new idea for the evaluation of blood injury. The standard Raman spectra of hemoglobin and red blood cells were collected and compared in order to determine the peak intensity of erythrocytes. The sheer force of 0, 50, 100, 150, 200, 250 and 300 pa was applied to the tested blood samples for 1 s on the blood shear test platform. Using confocal Raman instrument, the Raman spectra of erythrocytes under shear stress were collected under 10 times long focal lens, 532 nm laser light source wavelength, integration time 10 s, twice, and 2.5 mW. Through the normalization method to compare the changes of the Raman spectrum of red blood cells, evaluate the degree of red blood cell Sublethal damage, and use the curve fitting method to fit the characteristic peak and shear stress to verify the ability of Raman spectrum to evaluate the sub-injury of red blood cells. Comparing the standard Raman spectra of hemoglobin and red blood cells, it is found that the erythrocyte spectra can reflect the internal structure of hemoglobin. The results show that Raman spectroscopy can be used to distinguish the Sublethal damage erythrocytes under different shear stress, and it is inferred that the shear stress can pass through the cell membrane and affect the internal hemoglobin structure. With the increase of shear force, the left spectral line of 1 376 cm-1 increased. Obviously, the peak intensity of 1 549 and 1 604 cm-1 increased, and the vibration band of oxygen concentration marker band 10 at 1 639 cm-1 decreased. Among them, the peak strength of 1 549 cm-1 position is the high spin band of ferrous ion. Under the action of different shear forces, the difference of peak strength is the most obvious, which has an obvious positive linear relationship with the shear stress, and the fitting effect is good. Raman spectroscopy has the advantages of simple treatment, short time, simple operation and good reproducibility can accurately detect the subtle changes in the internal structure of cells, and evaluate the degree of sub-injury of red blood cells. it makes up for the deficiency of the traditional evaluation of hemolysis, provides a new technical means for the evaluation of blood injury caused by artificial heart pump, and broadens the application field of the Raman detection method.