| Laser tweezers Raman spectroscopy(LTRS) is a biological photonic technology that the optical trap technique combined with Raman spectroscopy and applied to study suspension cell. With a beam of light to achieve prison single suspension cell then stimulated the Raman spectra, LTRS is a new tool to study the organization of object molecular structure, also a popular tool in the biological field of study single-cell molecular level at present.Integrated and combined are the characteristics of microfluidic laboratory on chip. Detection is an important part of microfluidic laboratory on chip, at the same time, it is a key technologies of the microfluidic chip's rapid development. As a new field, microfluidic chip combines with chemical synthesis, biological analysis, optical and information science, which has brought a major impact. Microfluidic chip combining high sensitivity micro-detection system is develop into the current hot spots. At present, some teams have successfully combined microfluidic chip technology with laser tweezers Raman spectroscopy, and get a better signal to noise ratio. However, automation after the integrated of microfluidic chip and Raman tweezers is an exciting topic.This thesis base on the existing system of laser tweezers Raman spectroscopy (LTRS), explorat to LTRS system as microfluidic chip's high-sensitivity detection methods, combine microfluidic chip with the LTRS then integration. microfluidic chip - Raman Tweezers Automatic analysis biosensor system for molecular and cellular level, to provide fast, simple, accurate, and non-invasive micro-total analysis system (μ-TAS) for studying single cell's physiology and biochemistry, medical diagnosis, pharmacology, drug screening high throughput, similar to the role of flow cytometry to automatically detect cell components.In this thesis have six chapters. Chapter introduction, a brief introduction historical background, theory, application areas in biology and Research of Raman spectra, respectively, laser tweezers, LTRS and microfluidic chip.Chapter II Application of Raman optical tweezers to study white blood cells during inflammation. The results show that normal and inflammatory white blood cells'Raman spectra have significant differences, identified by spectra show that in the state of inflammation, protein amino acid content in leukocytes unchanged, but significant changes in protein structure; bases of nucleic acid are increase, DNA double-helix structure has change. The method of PCA principal component can completely distinguish the normal and inflammatory white blood cells.Chapter III Application STRS to analysis the material composition of Trichomonas vaginalis (TV) and Trichomonas tenax (TT), to find characteristic Raman peaks of taxonomic identification. Results show that they have different peak intensity and spectral type in 937 cm-1, 1002 cm-1 and 1446 cm-1. According to 1002 cm-1 peak of the signal strength, and combined with 937 cm-1 and 1002 cm-1 peak intensity ratio (I937/I1002) ,1002 cm-1, 1446 cm-1 peak intensity ratio (I1002/I1446) can serve as a quantitative indicators to distinction Trichomonas vaginalis and Trichomonas tenax. The above studys show that: laser tweezers Raman spectroscopy can be an effective means of single-cell research in biology.ChapterⅤAutomation Platform of microfluidic chip combine with LTRS. The study of Microfluidic chip system unit: includ the choice of chip substrate material, capillary and the method of making chip. Research of Microfluidic chip-LTRS automatic system unit: Design a solenoid valve controlling laser switches, use a electronic control scan mirror to achieve optical tweezers scanning, function signal generator control optical tweezers'amplitude and frequency, the efficiency of optical tweezers trapping cells by changing the laser power and liquid flow rate and fluid density, image recognition method use to judge whether optical tweezers has trap cell, laser and scanning switches are switches and output through the computer to control the high and low. Automation Control System: Using visual basic and visual c + + software editing program to achieve automate. Control processes: Sample flow --- Laser on --- optical tweezers scan --- blank test --- determine threshold --- capture detector --- confirm capture --- collect spectra ---- complete spectrum collection --- laser off --- elease cell --- Laser on.ChapterⅥthe application of microfluidic chip combine LTRS biosensor automation system in the single cell level. We measuremented the Raman spectroscopy of normal andβ-thalassemia red blood cells, the result show that in the condition of 3s integration time, 500 cells can be detected within one hour. Base on datas processing, found that the average Raman spectra between normal andβ-thalassemia red blood cells are different, characteristic peaks are 1004,1130,1450,1545,1604 and 1618 cm-1, these peaks are Attributable to protein ingredients. Each of these characteristic peaks of the normal picture out and carry out statistical analysis, the normal distribution chart shows thatβthalassemia single red blood cell's protein content distribution is symmetrical, the maximum ratio higher, the minimum is lower and the standard deviation larger than normal red blood cells. This shows that the material withinβ-thalassemia red blood cells is uniformity than normal red blood cells. Characteristic peaks of the statistical analysis show thatβ-thalassemia red blood cell's hemoglobin distribution wider than normal red blood cells, the average intensity difference of less than 6%. In order to determine the relationship of the individual cells better, we arbitrary pumping two groups of normal and thalassemia cells, each of 200 cells the shots component analysis, sensitivity is 93.5%, specificity is 99.5%, using the principal component analysis method can well distinguished normal andβ-thalassemia red blood cells, and from the load diagram of the principal component analysis it is obvious to see the changes. Collected 20 cells from 200 cells as test datas, another 180 cells to develop base failure, then the 20 cells have fallen into a certain area. Using the same method to several sets of data simultaneously and found that the same type of cells are gathered in one area, this unit has played a similar role of flow cytometry. Base on all these stringent data processing, can get the same results, it is better show the advantages of microfluidic chip - LTRS automated system.Chapter VI Conclusion and prospect. Summarizes this study, point out the significance and lacking, look into the future of this work.The above studies suggest that: LTRS and LTRS-microfluidic chip automation systems can play an important role in biology research, provide a reliable and convenient means to medical diagnosis research.
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