Analysis Of Hemoglobin In Single Red Blood Cells

Hemoglobin(Hb)is a protein responsible for carrying oxygen in red blood cells present in higher organisms,and it is the main component of red blood cells.Currently the concentration of hemoglobin is an important indicator that people are more concerned about,because it can not only evaluate the prevalence of anemia in the population,but also reflect the nutritional status of iron in the body.Thus a convenient and effective method of evaluating the hemoglobin concentration detection of human health is very important,especially in hemoglobin concentration at single cells level.In this thesis,the hemoglobin concentration of single red blood cells was analyzed rapidly and efficiently by mainly using Raman spectroscopy,meanwhile combined with UV spectrophotometric measurement and holographic imaging.These single cells analysis ways are of high-definition resolution,and are non-destructive.Hence the sensitivity and accuracy of detection are improved further,and it is possible to study single living cells in solution.As a matter of fact,three aspects of work were carried out as follows:1.Raman spectroscopy was employed to measure the hemoglobin concentration within a single red blood cell:First,we extracted the hemoglobin from red blood cells,and measured the Raman spectra of different concentrations of hemoglobin.Secondly the linear fitting of the Raman spectra intensity of peaks at 1546 cm-1 and hemoglobin concentration was made,and than we detected the Raman spectroscopy of 70 red blood cells.Thirdly by substituting the linear equation and we got each red blood cell hemoglobin concentration.As a result,the single red blood cell hemoglobin concentration is between 270?500 g/L,wherein the concentration of hemoglobin in the 350?450 g/L is mature red blood cell,more than 90%of the total,while the concentration of 300 g/L or less is likely reticulocytes,the concentration beyond 450 g/L or more is aging red blood cell.The results showed that the mean corpuscular hemoglobin concentration of 70 red blood cells is 369 g/L,which in line with mean corpuscular hemoglobin concentration(MCHC):310?370 g/L.At last we compared the blood cell of human with the chickens and mice blood cells,and then analyzed their hemoglobin content of individual red blood cells,obtained the differences of different kinds of biological cells.2.We illustrated a lensfree holographic on-chip imaging technology.By controlling the spatial coherence of the illumination source,we record a 2D holographic diffraction pattern of each cell or micro-particle on a chip using a high resolution sensor array.The recorded holographic image is then processed by using a custom developed decision algorithm for matching the detected hologram texture to existing library images for on-chip characterization,then get a single red blood cell hemoglobin concentration rapidly.The holographic diffraction signature of any microscopic object is significantly different from the classical diffraction pattern of the same object.It improves the signal to noise ratio and the signature uniformity of the cell patterns;and also exhibits much better sensitivity for on-chip imaging of weakly scattering phase objects such as cells.Compared to the existing imaging system on-chip,we improved the performance of the method by characterized automatic blood cell heterogeneous solution significantly.Combined with Raman spectroscopy technology,it can be quickly verified as a rapid,non-destructive,efficient new method to detect single red blood cell hemoglobin concentration in theory.3.By using laser tweezers Raman system and SERS,the Ag@AuNPs were injected into the mice to study the real-time process of the co culture of red blood cells and particles,the effect of the Ag@AuNPs on blood Raman spectra,and the metabolism of metal nanoparticles in vivo.By analyzing the characteristic peak of Raman spectrum,the results show that the intensity of the Raman characteristic peak of red blood cell in the Raman spectrum increases first and then decreases with the time.The feasibility of SERS in living organisms is proved.It is very important for understanding the metabolism of nanoparticles in animals and the use of nanoparticles for disease detection.