【作者】 吕弋；

【导师】 章竹君；

【作者基本信息】 西南师范大学 ， 分析化学， 2003， 博士

【摘要】 本工作的主要内容是基于微流控技术的化学发光生物传感器芯片以及新型化学发光体系的研究，全文由二部分组成。 第一部分研究了基于微流控技术的化学发光生物传感器芯片及其在临床上重要的生化物质（尿酸，葡萄糖）的分析应用。本文所提出的生物传感器芯片的最大特点是首次用空气替代传统的溶液作为试样驱动介质。在用各种检测手段的流动注射分析或流通式传感器中，毫无例外都是用溶液作为载流进行驱动，并且尽可能避免在流路中出现气泡，而本文提出的以空气作为载流的方法有以下优点：在微量和超微量分析中，所用溶液的背景信号，即由于化学干扰而产生的背景信号将是控制方法灵敏度的重要因素，特别是在生物传感器芯片上尤为重要，通常用溶液作为载流，这一背景是不可能避免的，而用空气作载流完全避免了这一化学干扰，提高了信噪比；在用溶液作为载流时，在未经处理的微通道中比较粗糙的管壁上经常会出现微气泡，且很难消除，它们是造成测定重现性降低的重要原因之一，而利用空气作为载流就不存在这一问题；在流动注射分析中，用载流驱动试样溶液时，样品带与载流之间的扩散使得样品带在流路中不断展宽，而用空气作为载流，样品带在流动过程中，将一直保持同样的宽度，从而提高测定的灵敏度。该部分主要包括四章，第一章主要回顾了生物传感器以及生物传感器芯片的产生，发展趋势及其应用。现代生物传感器正向着微型化和芯片化方向发展。 第二章研究了基于微流控技术及微型反应器的化学发光尿酸生物传感器芯片的设计。该传感器芯片大小仅为25mm×75mm×6.5mm，在实验室中通过现代微加工技术制备。该芯片由微型反应器，微型酶反应器以及微型流路组成，反应试剂鲁米诺和辣根过氧化酶（HRP）通过经典的Sol-gel方法固定于微反应器中，而尿酸氧化酶也通过该方法固定于微型酶反应器中。该传感器芯片的最大特点是首次用空气替代传统的溶液作为系统的载流。尿酸样品在酶反应器中产生的过氧化氢流经微反应器时，产生化学发光，据此实现对尿酸的测定。所研制的生物传感器芯片对尿酸的线性响应范围为1.0～100mg.L^-1(ΔI=3.09C(mg.L^-1)+2.1，r²=0.9992)。检测限为0.1mg.L^-1（3σ）。对浓度为50mg.L^-1尿酸溶液平行测定7次所得相对标准偏差为 4二％。该传感器芯片保存于冰箱（4 OC）10天后无明显变化，且能连续重复使用200次而无明显变化（测定值RSD小于5％），该传感器芯片成功用于血清中尿酸含量的测定，结果令人满意。 第三章研究了同样基于微流控技术以及微型反应器的化学发光葡萄糖传感器芯片。该芯片由发光试剂池，酶反应池，微反应混合器以及微型流路组成，发光试剂鲁米诺和铁氰化钾分别通阴离子交换树脂固定，然后按一定比例均匀混合于发光试剂储备池，葡萄糖氧化酶通过多孔玻璃（CPG）固定，并置于酶反应池中。尿酸样品经酶反应池产生的过氧化氢与从发光试剂中淋洗下来的鲁米诺和铁氰化钾在微反应混合器中反应并产生化学发光。所研制的传感器芯片对葡萄糖测定的线性范围为 1．1－110mmol．L’，其线性关系为Ahs刀gC（mmol．L‘）＋12．1仁’－0．9991，n－7)，检坝卜为 0．Immol．L‘（3）。对 5．5 mmol．L’葡萄糖平行狈定 7次，其相对标准偏差为3．9％。该传感器芯片能重复使用200次而无明显变化(测定值RSD小于5％人 该传感器芯片成功用于血清中葡萄糖的测定，结果令人满意。 第四章中，研究了鲁米诺－铁氰化钾－EDTA－甲基多巴这一化学发光反应特点，结果表明鲁米诺－铁氰化钾－甲基多巴在碱性条件下能产生强烈的化学发光，该反应为快发光过程，当一定量的EDTA存在下，该发光转变成一慢发光过程。据此，将该发光体系应用于化学发光成像分析测定甲基多巴的研究中。在一定的实验条件下，发光强度与甲基多巴浓度成良好的线性关系。该工作为化学发光阵列芯片测定药物的研究奠定了良好的基础。 近年来，包括基因药物、生物药物及化学药物在内的各类新型药物的制备。合成和筛选己成为药物研究的重要内容，在我国加入WTO后，已显得更加迫切。现有的分析手段许多己不能满足要求，在未来10—15年内阵列芯片和微流控芯片将担当主要的作用，而以化学发光为基础的微阵列和微流控芯片，由于具有高灵敏度、检测器易于微型化和廉价的特点将成为发展的主要方向之一，本文第二部分中一共对六种能应用于流动注射化学发光药物分析芯片的几个发光体系进行了研究，为进一步的芯片制作打下了基础。这些药物包括多酚，硫酸铁布他林，甲基多巴，利巴威林，富马酸同替芬以及奥硝哗。前三个药物是基于铁氰化钾－荧光染料（罗丹明B，罗丹明6G和二氯荧光素）化学发光体系进行测定的，而利巴威林，富马酸同替芬以及奥硝哇则分别利用的是鲁米诺．过硫酸钠，鲁米诺铁氰化钾体系测定。铁氰化钾－荧光染料化学发光体系属于首次报道，通过对该类体系的化学发光光谱的分析，发现该类体系的共同特点是，铁氰化钾氧化多酚类化合物，产生的能量转移给荧光染料，然后产生强烈发光。 第一章研究了铁?更多还原

【Abstract】 In the present work, the investigation on the biosensor chip with Chemiluminescence detection and novel Chemiluminescence systems is reported in part I and II, respectively.Part I. In this section, two biosensor chips coupled to microfluidic analysis system with Chemiluminescence detection are developed for the determination of clinically important biochemical substances including uric acid and glucose in serum. In general, for all the assays on chip with micromechanical pumping devices, the carrier solution was required. In the present work, we took the air as the carrier flow instead of solution on a biosensor for the first time. The distinguished possible advantages of this microfluidic system based on carrier airflow 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 affected the sensitivity. Especially in the present microfulidic system, the chemical background which was usually inevitable because of the carrier solution, would be eliminated by using air as the carrier flow, thus a large signal to noise ratio (SNR) and a large 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 eliminate and often affected 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 (obviously, the case in which the carrier solution was the reaction reagents was not included). However, when the air was used as the carrier flow instead of the solution, the phenomenon of spreading disappeared, and a great sensitivity could be attained. This part is divided into four chapters. Chapter 1 reviews the development of the biosensor and biosensorchip in recent years.In chapter 2, a chemiluminescence biosensor on a chip coupled to microfluidic analysis system and microreactor is described. The chemiluminescence biosensor measured 25+75+6.5mm in dimension, and was readily produced in analytical laboratory. The sol-gel method is introduced to co-immobilize horseradish peroxidase (HRP) and luminol in the microreactor, and to immobilize uricase in the enzymatic reactor. The main characteristic of the biosensor was to introduce the air as the carrier flow instead of the common solution carrier for the first time. The uric acid was sensed by the CL reaction between hydrogen peroxide produced from the enzymatic reactor and luminol under the catalysis of HRP in the microreactor. The present biosensor chip was successfully applied to the measurements of uric acid in serum. Under the optimum conditions, the response to the uric acid concentration was linear over range1.0 to 100 mg.L-1 with a regression equation of I=3.09C (mg.L-1) + 2.1 (r2=0.9992, n=6) and a detection limit of 0.1 mg.L-1(3). The relative standard deviation for 50 mg.L-1 uric acid was 4.2% (n=7). After the enzymatic reactor was hermetically stored at 4C for 10 days, no significant changes were observed in response characteristics of the system. More than 200 measurements were carried out during this time. The proposed biosensor chip was successfully applied to the measurement of uric acid in human serum.In chapter 3, a chemiluminescence biosensor on a chip coupled to microfluidic system is described in this paper. The chemiluminescence biosensor measured 25+45+5mm in dimension, was readily produced in analytical laboratory. Glucose oxidase (GOD) was immobilized onto controlled-pore glass (CPG) via glutaraldehyde activation and packed into a reservior. 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 instead更多还原