本文關(guān)鍵詞：血液分離微流控SERS芯片的設(shè)計制備及其應(yīng)用研究　出處：《重慶大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 血液 微流控SERS芯片 Ag film@nano Au 肌酐

【摘要】：微流控芯片具有較好的生物相容性,并且有著樣本需求量少,產(chǎn)生廢棄物少,反應(yīng)速度快,環(huán)境友好等獨特的優(yōu)勢。同時,表面增強拉曼光譜(SERS)檢測技術(shù)靈敏度高,不需要復(fù)雜的樣本預(yù)處理,與微流控芯片具有很好的兼容性,在血液分析領(lǐng)域引起廣泛的關(guān)注。但目前針對血液樣本的微流控SERS分析檢測一體化方面的研究還比較少。其根本原因在于血液成分比較復(fù)雜,各物質(zhì)之間的特征峰可能發(fā)生重疊,因此需要SERS信號重現(xiàn)性好和靈敏度高的SERS基底。若能獲得適用于血液成分檢測的高重現(xiàn)性和高靈敏度的SERS基底,將其集成在微流控芯片中,將有利于減少血液樣本的用量,實現(xiàn)血液成分的分離檢測一體化,并有利于實現(xiàn)血液相關(guān)疾病的快速診斷識別。本文的主要研究內(nèi)容及其結(jié)果如下:(1)設(shè)計了血液分離微流控芯片,實現(xiàn)了血細(xì)胞和血清的有效分離,成功開展了血細(xì)胞和血清的普通Raman測試分析。根據(jù)血細(xì)胞和血清密度之間差異較大,因此相同條件下所受到的離心力作用不同的原理,設(shè)計出由分離-傾析結(jié)構(gòu)、混合管道和SERS檢測區(qū)構(gòu)成的圓形PDMS-玻璃微流控芯片。8個單元結(jié)構(gòu)兩兩對稱分布,分離-傾析結(jié)構(gòu)位于遠(yuǎn)離圓心的位置,有利于增強離心力的作用。分離腔和傾析腔之間通道的垂直距離X分別為1mm、2mm、3mm,在1000rpm離心轉(zhuǎn)速下持續(xù)90s,X=2mm時血清-血細(xì)胞的分離傾析效果最好。通過對分離得到的血細(xì)胞進(jìn)行原位Raman測試,結(jié)果顯示,相比于開敞體系血細(xì)胞的Raman特征峰,微流控芯片中的血細(xì)胞背景熒光造成的基線漂移得到了明顯的降低。并且,經(jīng)過分離濃縮的血細(xì)胞Raman特征峰更為豐富,峰強更強,將有利于血細(xì)胞特征峰的歸屬識別。對15例健康人和20例慢性腎衰竭患者的血細(xì)胞Raman譜圖進(jìn)行分析,結(jié)果顯示,健康人位于756 cm-1、1004 cm-1、1122 cm-1、1226 cm-1、1550 cm-1、1640cm-1處譜線相對于1618cm-1處絡(luò)氨酸C=C振動特征峰強度均比慢性腎衰患者強,與多數(shù)慢性腎衰竭患者均伴有不同程度的貧血的現(xiàn)象是一致的。并且1004 cm-1處健康人譜線強度比慢性腎衰竭患者略強,說明慢性腎衰減患者血細(xì)胞中苯丙氨酸的含量略低于健康人。而普通Raman測試不能獲得血清的有效Raman信息。(2)基于金和銀優(yōu)異的等離子特性,設(shè)計了Ag film@nano Au新型SERS基質(zhì),成功地將其原位集成在“三明治”式微流控SERS芯片中,并開展了血清SERS測試。采用自組裝-化學(xué)鍍法將Ag film@nanoAu SERS基質(zhì)集成在簡易“三明治”微流控芯片中,制備得到“三明治”微流控SERS芯片。采用SERS常用探針分子R6G,對Ag film@nano Au SERS基質(zhì)的制備條件進(jìn)行了實驗優(yōu)化,并對制備得到的微流控SERS芯片進(jìn)行了效能分析和血清SERS測試分析。PDDA濃度為0.01%,化學(xué)鍍時間為4min時,該微流控SERS芯片的SERS增強效果最佳。在最佳條件下制備得到的Ag film@nano Au SERS基質(zhì),相比于單一的金納米粒子膜和銀納米膜,R6G位于1507cm-1處C-C伸縮振動峰的SERS信號強度提高了3-5倍。測試時,當(dāng)激光聚焦在待測物分子與SERS基質(zhì)的界面上時可獲得最強的SERS信號;相同厚度的PDMS蓋片和玻璃蓋片對R6G的SERS信號強度分別衰減2.5倍和2.1倍。采用1mm厚的PDMS作蓋片,該微流控SERS芯片依然可實現(xiàn)10nM R6G的檢測,增強因子達(dá)3.8×105;不同批次制備的微流控SERS芯片獲得的R6G的SERS信號的RSD低至10%左右。將該微流控SERS芯片應(yīng)用于人血清的檢測,血清的特征峰得到了明顯的增強,說明該微流控芯片可用于血清及血清成分的SERS測試研究。(3)基于集成了Ag film@nano Au SERS基質(zhì)的微流控SERS芯片,開展了血清中肌酐的SERS測試分析,實現(xiàn)了慢性腎衰竭病人血清中肌酐含量范圍的初步判定。自組裝-化學(xué)鍍法制備的微流控SERS芯片對肌酐水溶液的檢測限為5×10-3mg/dl。將其用于血清中肌酐的SERS測試分析,血清中肌酐濃度為20.0-20.5mg/dl依然不能獲得肌酐的有效SERS信號。采用自組裝法對微流控芯片中Agfilm@nano Au SERS基底的制備條件進(jìn)行實驗優(yōu)化,提高對肌酐的檢測靈敏度。當(dāng)PDDA濃度為1%,金納米粒子粒徑約為10nm時,改進(jìn)后的Ag film@nano Au SERS基底的SERS增強效果最佳。對肌酐的檢測限達(dá)到5×10-5mg/dl,比自組裝-化學(xué)鍍法制備得到的微流控SERS芯片降低了兩個數(shù)量級。采用改進(jìn)后的微流控SERS芯片對人血清中肌酐進(jìn)行加標(biāo)測試,當(dāng)血清中肌酐濃度≥10.0mg/dl時,肌酐位于678cm-1處的特征峰可以被明顯識別,而當(dāng)血清中肌酐濃度10.0mg/dl時,不能觀察到肌酐的位于678cm-1處的特征峰。將改進(jìn)后的Ag film@nano Au SERS基底集成在血液分離微流控芯片的檢測區(qū),對典型慢性腎衰竭病人血液分離,在檢測區(qū)對分離得到的血清進(jìn)行SERS測試,整個過程2min。測試結(jié)果顯示,SERS測試所得血清中肌酐含量范圍,與酶法測試數(shù)據(jù)一致。本文設(shè)計制備的血液分離微流控SERS芯片可實現(xiàn)血液分離和血清中肌酐SERS一體化快速分析測試。
[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.
【學(xué)位授予單位】：重慶大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：O657.37;R446

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 陶琴;董健;錢衛(wèi)平;;表面增強拉曼光譜在定量分析中的應(yīng)用[J];化學(xué)進(jìn)展;2013年06期

2 孟令晶;林漫漫;牛麗媛;劉軍賢;王何健;姚輝璐;;活體小鼠中單個紅細(xì)胞的拉曼光譜分析[J];分析化學(xué);2011年09期

3 崔顏;任斌;田中群;;基于納米技術(shù)的表面增強拉曼光譜在細(xì)胞研究中的應(yīng)用[J];東南大學(xué)學(xué)報(醫(yī)學(xué)版);2011年01期

4 程海平;廖璞;;血清中肌酐的檢測方法及其進(jìn)展[J];檢驗醫(yī)學(xué)與臨床;2009年19期

相關(guān)碩士學(xué)位論文 前1條

1 楊艷;血液的Raman光譜和熒光光譜幾種新現(xiàn)象研究[D];南京理工大學(xué);2006年

，

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