血液分离微流控SERS芯片的设计制备及其应用研究

发布时间：2017-12-27 01:22

本文关键词：血液分离微流控SERS芯片的设计制备及其应用研究　出处：《重庆大学》2016年硕士论文　论文类型：学位论文

更多相关文章：血液 微流控SERS芯片 Ag film@nano Au 肌酐

【摘要】：微流控芯片具有较好的生物相容性,并且有着样本需求量少,产生废弃物少,反应速度快,环境友好等独特的优势。同时,表面增强拉曼光谱(SERS)检测技术灵敏度高,不需要复杂的样本预处理,与微流控芯片具有很好的兼容性,在血液分析领域引起广泛的关注。但目前针对血液样本的微流控SERS分析检测一体化方面的研究还比较少。其根本原因在于血液成分比较复杂,各物质之间的特征峰可能发生重叠,因此需要SERS信号重现性好和灵敏度高的SERS基底。若能获得适用于血液成分检测的高重现性和高灵敏度的SERS基底,将其集成在微流控芯片中,将有利于减少血液样本的用量,实现血液成分的分离检测一体化,并有利于实现血液相关疾病的快速诊断识别。本文的主要研究内容及其结果如下:(1)设计了血液分离微流控芯片,实现了血细胞和血清的有效分离,成功开展了血细胞和血清的普通Raman测试分析。根据血细胞和血清密度之间差异较大,因此相同条件下所受到的离心力作用不同的原理,设计出由分离-倾析结构、混合管道和SERS检测区构成的圆形PDMS-玻璃微流控芯片。8个单元结构两两对称分布,分离-倾析结构位于远离圆心的位置,有利于增强离心力的作用。分离腔和倾析腔之间通道的垂直距离X分别为1mm、2mm、3mm,在1000rpm离心转速下持续90s,X=2mm时血清-血细胞的分离倾析效果最好。通过对分离得到的血细胞进行原位Raman测试,结果显示,相比于开敞体系血细胞的Raman特征峰,微流控芯片中的血细胞背景荧光造成的基线漂移得到了明显的降低。并且,经过分离浓缩的血细胞Raman特征峰更为丰富,峰强更强,将有利于血细胞特征峰的归属识别。对15例健康人和20例慢性肾衰竭患者的血细胞Raman谱图进行分析,结果显示,健康人位于756 cm-1、1004 cm-1、1122 cm-1、1226 cm-1、1550 cm-1、1640cm-1处谱线相对于1618cm-1处络氨酸C=C振动特征峰强度均比慢性肾衰患者强,与多数慢性肾衰竭患者均伴有不同程度的贫血的现象是一致的。并且1004 cm-1处健康人谱线强度比慢性肾衰竭患者略强,说明慢性肾衰减患者血细胞中苯丙氨酸的含量略低于健康人。而普通Raman测试不能获得血清的有效Raman信息。(2)基于金和银优异的等离子特性,设计了Ag film@nano Au新型SERS基质,成功地将其原位集成在“三明治”式微流控SERS芯片中,并开展了血清SERS测试。采用自组装-化学镀法将Ag film@nanoAu SERS基质集成在简易“三明治”微流控芯片中,制备得到“三明治”微流控SERS芯片。采用SERS常用探针分子R6G,对Ag film@nano Au SERS基质的制备条件进行了实验优化,并对制备得到的微流控SERS芯片进行了效能分析和血清SERS测试分析。PDDA浓度为0.01%,化学镀时间为4min时,该微流控SERS芯片的SERS增强效果最佳。在最佳条件下制备得到的Ag film@nano Au SERS基质,相比于单一的金纳米粒子膜和银纳米膜,R6G位于1507cm-1处C-C伸缩振动峰的SERS信号强度提高了3-5倍。测试时,当激光聚焦在待测物分子与SERS基质的界面上时可获得最强的SERS信号;相同厚度的PDMS盖片和玻璃盖片对R6G的SERS信号强度分别衰减2.5倍和2.1倍。采用1mm厚的PDMS作盖片,该微流控SERS芯片依然可实现10nM R6G的检测,增强因子达3.8×105;不同批次制备的微流控SERS芯片获得的R6G的SERS信号的RSD低至10%左右。将该微流控SERS芯片应用于人血清的检测,血清的特征峰得到了明显的增强,说明该微流控芯片可用于血清及血清成分的SERS测试研究。(3)基于集成了Ag film@nano Au SERS基质的微流控SERS芯片,开展了血清中肌酐的SERS测试分析,实现了慢性肾衰竭病人血清中肌酐含量范围的初步判定。自组装-化学镀法制备的微流控SERS芯片对肌酐水溶液的检测限为5×10-3mg/dl。将其用于血清中肌酐的SERS测试分析,血清中肌酐浓度为20.0-20.5mg/dl依然不能获得肌酐的有效SERS信号。采用自组装法对微流控芯片中Agfilm@nano Au SERS基底的制备条件进行实验优化,提高对肌酐的检测灵敏度。当PDDA浓度为1%,金纳米粒子粒径约为10nm时,改进后的Ag film@nano Au SERS基底的SERS增强效果最佳。对肌酐的检测限达到5×10-5mg/dl,比自组装-化学镀法制备得到的微流控SERS芯片降低了两个数量级。采用改进后的微流控SERS芯片对人血清中肌酐进行加标测试,当血清中肌酐浓度≥10.0mg/dl时,肌酐位于678cm-1处的特征峰可以被明显识别,而当血清中肌酐浓度10.0mg/dl时,不能观察到肌酐的位于678cm-1处的特征峰。将改进后的Ag film@nano Au SERS基底集成在血液分离微流控芯片的检测区,对典型慢性肾衰竭病人血液分离,在检测区对分离得到的血清进行SERS测试,整个过程2min。测试结果显示,SERS测试所得血清中肌酐含量范围,与酶法测试数据一致。本文设计制备的血液分离微流控SERS芯片可实现血液分离和血清中肌酐SERS一体化快速分析测试。
[Abstract]:Microfluidic chip has good biocompatibility, and has the unique advantages of less sample demand, less waste, fast reaction speed, environment friendly and so on. At the same time, surface enhanced Raman spectroscopy (SERS) detection technology is highly sensitive, and does not require complex sample pretreatment. It has good compatibility with microfluidic chips, and has attracted wide attention in the field of blood analysis. However, there are few studies on the integration of microfluidic SERS analysis and detection for blood samples. The fundamental reason is that the blood components are complex, and the characteristic peaks of all substances may overlap. Therefore, SERS substrates with good reproducibility and sensitivity for SERS signals are needed. If you can get for blood component detection of high reproducibility and high sensitivity of the SERS substrate, which is integrated in a microfluidic chip, will help reduce the amount of blood sample, realize the integration separation and detection of blood components, and is conducive to the realization of the rapid diagnosis of blood related diseases. The main contents and results of this paper are as follows: (1) designed a blood separation microfluidic chip to achieve effective separation of blood cells and serum, and successfully carried out routine Raman test analysis of blood cells and serum. According to the great difference between blood cell and serum density, so the principle of centrifugal force under the same conditions is different. A circular PDMS- glass microfluidic chip made up of separation and decanting structure, mixed pipeline and SERS detection area is designed. The 8 element structure is 22 symmetrical distribution, and the separation - dipping structure is located far away from the center of the center, which is beneficial to the enhancement of the centrifugal force. The vertical distance between the separation chamber and the decanting chamber is X, 1mm, 2mm and 3mm, respectively, and the best effect is the separation and decanting of serum and blood cells at 1000rpm centrifugal speed. The in situ Raman test of isolated blood cells showed that compared with the Raman characteristic peak of blood cells in open system, baseline drift of blood cell background fluorescence in microfluidic chips was significantly reduced. Moreover, the Raman characteristic peaks of the isolated blood cells are more abundant and stronger, which will be beneficial to the identification of the characteristic peaks of blood cells. The blood Raman cells of 15 healthy people and 20 cases of chronic renal failure patients with spectrum analysis, results showed that healthy people at 756 cm-1, 1004 cm-1, 1122 cm-1, 1226 cm-1, 1550 cm-1, 1640cm-1 line relative to 1618cm-1 tyrosine C=C vibration peak intensity were higher than in patients with chronic renal failure. It is consistent with the majority of patients with chronic renal failure were associated with varying degrees of anemia phenomenon. At 1004 cm-1, the intensity of healthy subjects was slightly stronger than that of patients with chronic renal failure, indicating that the level of phenylalanine in blood cells of patients with chronic renal failure is slightly lower than that of healthy people. The common Raman test could not obtain the effective Raman information of the serum. (2) based on the excellent plasma characteristics of gold and silver, a new Ag film@nano Au SERS matrix was designed, which was successfully integrated into the sandwich type microfluidic SERS chip successfully, and the serum SERS test was carried out. The Ag film@nanoAu SERS substrate was integrated into a simple sandwich microfluidic chip by self-assembly electroless plating, and the sandwich microfluidic SERS chip was prepared. The preparation conditions of Ag film@nano Au SERS substrate were optimized by using SERS commonly used probe molecule R6G, and the efficiency analysis and serum SERS test analysis of the prepared microfluidic SERS chip were made. When the concentration of PDDA is 0.01% and the time of electroless plating is 4min, the SERS enhancement effect of the microfluidic SERS chip is the best. Under the optimum conditions, the Ag film@nano Au SERS matrix was prepared, and the SERS signal intensity of C-C stretching vibration peak at R6G was 3-5 times higher than that of single gold nanoparticle film and silver nano film. When testing, when laser is focused on the interface between the tested molecules and the SERS matrix, the strongest SERS signal can be obtained. The PDMS signal intensity of the same thickness of the PDMS cover and the glass cover slice is 2.5 times and 2.1 times of the SERS signal intensity, respectively. Using 1mm thick PDMS as cover, the microfluidic SERS chip still can detect 10nM R6G, and the enhancement factor is 3.8 x 105. RSD of SERS signal obtained from different microfluidic chips prepared by different batches is as low as 10%. The microfluidic chip SERS has been applied to the detection of human serum and the characteristic peak of serum has been significantly enhanced, indicating that the microfluidic chip can be used for SERS test of serum and serum components. (3) based on the microfluidic SERS chip integrated with Ag film@nano Au SERS matrix, we carried out the SERS test and analysis of serum creatinine, and achieved the preliminary judgement of the serum creatinine content in patients with chronic renal failure. The detection limit of microfluidic SERS chips prepared by self assembly and electroless plating is 5 x 10-3mg/dl for creatinine solution. The SERS test was used to test the serum creatinine in serum. The serum creatinine concentration of 20.0-20.5mg/dl still failed to obtain the effective SERS signal of creatinine. The self-assembly method was used to optimize the preparation conditions of Agfilm@nano Au SERS substrate in microfluidic chip, and improve the sensitivity of creatinine detection. When the concentration of PDDA is 1% and the particle size of gold nanoparticles is about 10nm, the improved SERS enhancement effect of the modified Ag film@nano Au SERS substrate is the best. The detection limit for creatinine is 5 * 10-5mg/dl, and two orders of magnitude lower than the microfluidic SERS chip prepared by self assembly and electroless plating. The microfluidic SERS chip after the improvement of creatinine in human serum by standard addition test, when the serum creatinine concentration greater than or equal to 10.0mg/dl, the characteristic peak of creatinine is located at 678cm-1 can be clearly identified, and when the 10.0mg/dl concentration of creatinine in serum, to observe a peak is located at 678cm-1 features of creatinine. The improved Ag film@nano Au SERS substrate was integrated in the detection area of the blood separation microchip, and the blood was separated from the typical chronic renal failure patients.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.37;R446

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