【作者】 陈福南；

【导师】 章竹君；

【作者基本信息】 西南师范大学 ， 分析化学， 2002， 硕士

【摘要】 本论文分为三部分。每个部分都包括相关综述和研究报告。 第一部分先介绍了微全分析系统的概况以及微流控芯片近年的研究状况，包括芯片的微细加工新技术和检测系统研究进展。微流控芯片是以分析化学为基础，以微机电加工为依托，以微管道网络为结构特征，以生命科学为主要应用对象，集成采样、稀释、加试剂、反应、分离、检测于一微芯片上的分析测试系统。其特点是大大降低试剂及试样消耗量，提高分析速度，减少测试费用。经过十多年的研究，微流控芯片在加工制作技术、材料选择、检测技术等方面都取得了极大的进展，为普及分析测试技术、实现分析实验室的家庭化、个人化创造了条件。在这部分的研究报告里，本文首次报道了用空气作载流的微流控芯片——化学发光检测系统。在微量分析和超微量分析里，化学干扰产生的溶液背景是影响灵敏度的主要因素，尤其在微流控系统中，载流溶液的背景不可避免，用空气作载流就可以消除背景而获得高信噪比和高灵敏度。其次，微流控系统中用溶液作载流，很难消除因微通道内壁粗糙而产生的气泡，常常影响稳定性和重现性。空气作载流则可避免这一缺陷。最后，流动注射分析中载流溶液与样品的相互扩散会导致样品区变宽而降低灵敏度。用空气作载流就没有扩散，灵敏度极高。 很显然，微流控系统中用空气作载流的分析方法不适于分光检测、电化学检测以及荧光检测，但用化学发光检测时则显示出独特的优势。迄今为止，这种方法未见报道，将会成为提高微流控分析系统灵敏度的重要技术。 我们在25×75×5mm的透明玻璃上用打孔器制作了两个通道作为酶柱和化学发光试剂柱。用sol-gel技术固定辣根过氧化物酶(HRP)和鲁米诺在微反应器里，而把尿酸酶固定在酶反应器中。通过测量微反应器里产生的过氧化氢与在酶反应器里的鲁米诺在HRP存在时发生的化学发光反应信号对尿酸进行传感。选好的条件下，尿酸浓度响应的线性范围为1-100mg／L，回归方程式为I=3.09C(mg／L)+2.1(r~2=0.9992，n=6)，检出限为0.1mg／L(3σ)。50mg／L的尿酸的相对标准偏差为4.2％(n=7)。 对葡萄糖传感器芯片的研究也是在自制的芯片上进行。酶柱里装入固定 了葡萄糖氧化酶的多孔玻璃，化学发光试剂柱里装吸附了！uminol和铁氰化钾 的离于交换树脂。两通道之间刻蚀了一条微通道相接。样品注入酶柱，使酶催 化葡萄糖氧化，把洗脱液（Na少OoNaOH）注入固定化化学发光试剂柱，启 动泵，酶柱中产生的HZOZ与离子交换树脂柱洗脱的CL试剂相遇在微反应池 里充分混合，产生CL信号。葡萄糖的浓度由化学发光强度而定。我们研究了 固定化反应器中酶反应的条件、洗脱液的pH值、空气载流流速对芯片性能的 影响，在最佳条件下，测量葡萄糖浓度的响应线形范围为 1．ill omM，回归 方程为I－2．09C（mM）＋12二l（r‘－0．9991，n＝7），检狈限为0．lmM（3a），对5．smM的 葡萄糖溶液的相对标准偏差为3．9％…－7）。固定了鲁米诺和铁氰化钾的柱子可 重复使用200次以上，系统性能无明显改变。两个月内进行了200次测量。 第二部分综述了光学式化学发光生物传感器的研究进展。光学式传感器 是光谱法中最活跃和最重要的研究领域，是将具有分子识别作用和换能作用 的固定化试剂染料、酶、辅酶、生物受体、抗原。抗体、核核酸、DNA、动 植物组织或细胞、微生物等的敏感膜以某种方式固定，对样品中的待测物质 进行选择性的分子识别、再转换成各种光信息，如紫外、可见及红外光的吸 收和反射、荧光、磷光、化学发光和生物发光、拉曼散射、光声和表面等离 子体共振等信号输出。光学式化学传感器具有很高的传输信息容量，可以同 时反映出多元成分的多维信息，并通过波长、相位、衰减、偏振和强度调制、 时间分辨、搜集瞬时信息来加以分辨，真正实现多道光谱分析和复合传感器 阵列的设计，达到复杂混合物中特定分析对象的检测。光学式化学传感器还 具有很好的电绝缘性，检测安全，测定的信号更加稳定，抗电磁干扰性能强， 对恶劣环境的适应性好。由于光学式化学传感器具有诸多优点，其应用领域 也不断扩大，必将对新的传感技术的发展作出极大贡献。这一部分的研究报 告主要描述了一种新的固定化试剂流动注射－化学发光传感器测定扑热息痛的 方法。分析试剂鲁米诺和铁氰化物被静电吸附固定在离子交换树脂柱上。用 磷酸盐从柱上洗脱出的鲁米诺和铁氰化物反应发生的信号在扑热息痛的存在 下会减弱。该传感器在 5刀xlo’乙 l刀xlo”飞／ml范围内对扑热息痛的浓度响应 成线性。检测限为4．7xlo”gg／ml＊a卜该传感器可稳定使用200次，成功地用于 扑热息痛药片的测定。更多还原

【Abstract】 The thesis consists of three parts. Each part includes a review and a reseach report.The first review introduces the development of the Miniaturized Total Analysis System(u-TAS) and the microfluidic analytical systems, including microfabrication techniques and detection technologies of microfluidic chips. In this paper, we developed a novel chemiluminescence biosensor chip for determination of uric acid in human serum based on microfluidic system and microreactor using air as a carrier flow. On this chip, uric acid was injected into the enzymatic reactor in which the uricase had already been immobilized by sol-gel, produced H2O2 was carried through the microchannel by the carrier flow (air) to the microreactor in which the luminol and HRP had already immobilized by sol-gel, then produced a CL signal. The distinguished advantages of this microfluidic system based on carrier air flow were shown in the next.Firstly, in microanalysis and ultra microanalysis. the solution background that resulted from the chemical interference, would be the primary factor that effected the sensitivity. Especially in the present microfluidic system, the chemical background which was usually inevitable because of the carrier solution, would be eliminated by means of using air as the carrier flow, thus a great signal noise ratio (SNR) and a great sensitivity could be obtained. Secondly, when solution was used as carrier flow in microfluidic system, small air bubbles taking shape in the rough inner wall of the microchannels were difficult to be eliminated and often effected the stability and the repeatability. Fortunately, the air carrier could avoid that disadvantage.Finally, the mutual spreading between the sample zone and the carrier solution in Flow Injection Analysis (FIA), would always result in the sample zone’s widening and reducing the sensitivity. However, when the air was used as the carrier flow instead of the solution, the phenomenon of spreading disappeared,and a great sensitivity was attained.It was obvious that the proposed analytical method of the air carrier in microfluidic system were not suitable for the spectrophotometric, electrochemical and fluorescence detection. However, when applied in chemiluminescence detection, the distinguished advantages of this method could be shown enough. This method has not been reported up to the present, and would be the important technique to increase the sensitivity of the microfluidic analysis system in the future.The chemiluminescence biosensor measured 25x75 x5mm in dimension, was readily produced using common aiguille in analytical laboratory. Under the selected conditions, the response to the uric acid concentration was linear over range 1 to 100 mg/L with a regression equation of 7=3.09C (mg/L) + 2.1 (r2=0.9992,n=6) and a detection limit of 0.1 mg/L (3a). The relative standard deviation for 50 mg/L uric acid was 4.2% (n=7).Glucose oxidase (GOD) was immobilized onto controlled-pore glass (CPG) via glutaraldehyde activation and packed into a channel. The analytical reagents, including luminol and ferricyanide, were electrostatically co-immobilized on an anion-exchange resin. The most characteristic of the biosensor was to introduce the air as the carrier flow in stead of the common solution carrier for the first. The glucose was sensed by the CL reaction between hydrogen peroxide produced from the enzymatic reaction and CL reagents, which were released from the anion-exchange resin. The proposed method has been successfully applied to the determination of glucose in human serum. The linear range of the glucose concentration was 1.1 to 110 mM and the detection limit was 0. ImM (3a).The second review is about the development of fiber optical sensors. A novel flow-injection chemiluminescence (CL) biosensor for determination of paracetamol is described in this paper. The analytical reagents, luminol and ferricyanide. were electrostatically coimmobilized on an anion-exchange resin column. The signal produced by the reaction between luminol and ferricyanide, wh更多还原