【作者】 林鹏；

【导师】 许金钩；

【作者基本信息】 厦门大学 ， 分析化学， 2002， 博士

【摘要】 采用环境敏感高分子作为载体通过均相反应、异相分离进行荧光免疫分析（FIA），以及应用稀土螯合物作为标记物进行时间分辨荧光免疫分析（TRFIA）是现代免疫分析方法中两个引人注目的领域。本论文选题于这两个研究方向，并开展相关研究工作。全文共分六章，主要包括环境敏感高分子的制备及在FIA中的应用和稀土螯合剂的合成与应用。 第一章从免疫分析的定义着手，明确了FIA的唯一共同特征，介绍了FIA的基本原理、测定形式及存在问题，从均相FIA、异相FIA以及均相反应、异相分离的FIA三方面综述了近来的进展，总结了FIA与信号放大、高效分离、自动化等技术的联用情况，在此基础上概括了今后FIA的发展趋势，最后提出了论文设想。 第二章合成了一种新型快速响应的温度敏感高分子聚（N-异丙基丙烯酰胺-丙烯酰胺）[P（NIP-AA）]，通过改变丙烯酰胺（AA）的含量来改变高分子的临界溶解温度（LCST），使之适用于不同用途。其中，LCST在37℃的温度敏感高分子可用于免疫分析，与聚-N-异丙基丙烯酰胺（PNIP）作载体相比，灵敏度相当，但由于相转变温度(T_tr)的提高，使得免疫反应的温度更接近于生物体的生理温度，并使免疫反应速率得到提高。以P（NIP-AA）做载体，建立了兔IgG的夹心型热敏相分离荧光免疫分析和人IgG的竞争型酶联荧光免疫分析，结果令人满意。 第三章首先论述了pH敏感高分子作为免疫分析载体相比于温度敏感高分子所具有的优越性，然后将N-异丙基丙烯酰胺（NIP）和甲基丙烯酸（MAA）共聚，合成了37℃下相转变pH值在5.6左右且性能优良的pH敏感高分子P（NIP-MAA），将其作为免疫分析的载体，建立了兔血清中兔IgG的测定。该法可在37℃进行免疫反应，反应平衡时间只需4min。简要阐明了pH敏感的机理并评价了抗体在pH敏感高分子上的固定效率和非特异吸附情况。实验发现采用热引发方式代替氧化还原引发来固定抗原或抗体，可提高固定化效率，固 ％8 定物质的兔疫活性也得到了改善，且其相转变PH更为狭窄。更重要的是：采 用这种热引发方式聚合使得一些必须在有机相或高温条件下合成的环境敏感高 分子也可适用于活性物质的固定。通过人 IgG的测定，将两种固定方式做了对 比，指出了两种方法的优缺点。将少量疏水性单体甲基丙烯酸丁酯（BWh）与 NIP和 MAA在有机相中和活性酯一起共聚，发现新合成的 PH敏感高分子临界 溶解PH（LCSP）提高到了6．0，更适合于免疫分析，利用竞争法进行了兔血清中 兔 lgG的测定。 第四章将NIP与N，N’－二甲基氨丙基甲基丙烯酚胺（DMAP）共聚，合成 出37叩下相转变PH值在7．4左右的PH敏感高分于P（NIP－DMAPM），当溶液PH在7．4以上时，高分子很快从溶液中沉淀出来，离心即可将沉淀与溶液分离。在7．4以下时，高分子又重新溶解，呈透明溶液。将其作为免疫分析的载体，建立了兔IPG和人IPG的PH敏感相分离荧光免疫分析法，由于其LCSP在中性，分离时对抗原一抗体复合物造成的损坏大为降低，反应温度的提高使得反应平衡时间也大为减少，并探讨了标记抗体在P（NIP－DMAPM）上的非特异吸附及其消除。 第五章合成了一种新型的荧光鳌合剂2，9－双［N，N－双（搂甲基）氨甲基卜1，10－（菲咯琳）（BBCAP），研究了其与稀土离于所形成的荧光螫合物探针的性能，该鳌合剂有着良好的水溶性，本身即可与铬、锨等稀土金属离子形成强荧光的鳌合物，不需要加入增强液。且与稀土离于所形成的螫合物荧光强度非常稳定，Tb士BCAP鳌合物标记的抗体稀释至 5 X 10叫‘’m几仍可加以测定，检测灵敏度大于一般的稀土类鳌合物标记抗体。此外，该探针发射波长在547urn，可有效避开生物背景样品的干扰（生物样品发射波长一般在320一470urn），更重要的是由于其荧光寿命较长，可望用在时间分辨荧光免疫分析中，使灵敏度得到进一步提高。我们将朋“P上的竣基通过碳化二亚胺与羊抗兔IgG抗体上的氨基偶联，进行标记，将兔IgG固定在温度敏感高分子PNIP上，建立了竞争型热敏相分离测定兔 IgG的新方法。 11 摘要 第六章用新型鳌合剂BBCAP测定铭和锨。稀土元素化学性质很相似，因 此，由于其它稀土元素的干扰，难于从它们的混合物中单独测定某一种元素。 荧光分析法因其高度的灵敏性和选择性引起了科学家的关注，但是，用于单～ 稀土金属离子的测定时，其选择性和灵敏度并没有真正得到解决。选用BBCAP 作为测定镐和锨的新型荧光试剂有望在选择性方面得到较大改善。实验结果表－明：测定锨时，其它稀土离子在摩尔比为 100时不干扰测定；测定铬时，除锨 以外其它稀土离子在摩尔比至少为 100时不干扰测定，只有锨的存在会增强镐 的荧光，但采用标准加入法或在相同的基质中测定可以消除至少1 倍锨的干 扰?更多还原

【Abstract】 The fluorescence immunoassay (FIA) based on a fast homogeneous antigen-antibody immune complexation reaction and a simple heterogeneous separation process by environment-sensitive polymer as a carrier is of great interest in recent years. The development of time-resolved fluorescence immunoassay (TRFIA) using rare-earth chelate as a label has been an attractive area in last decade. The main efforts of this dissertation are thus centered on these two research directions. The dissertation consists of six chapters, which basically involves (1) preparation of environment-sensitive phase separating polymer and its application in FIA; (2) synthesis of rare-earth chelating agent and its application.In chapter 1, the basic principle, test formats and problems in FIA were introduced. The recent advances in homogeneous FIA, heterogeneous FIA, FIA with homogeneous reaction and heterogeneous separation were reviewed. The combination of FIA with amplification of fluorescence signal, high efficient separation and automatic technique was summarized. The research proposal for this dissertation was also presented.In chapter 2, A novel temperature-sensitive polymer, poly(N-isopropylacrylamide-acrylamide) [P(NIP-AA)J, was synthesized. Its lower critical solution temperature (LCST) could be regulated by changing the content of acrylamide (AA) for different purposes. The P(NIP-AA) with LCST of 37癈 was used as a separating carrier for the reactants in immunoassay. The sensitivity of this method was comparable to that using poly-N-isopropylacrylamide(PNIP) as a carrier. However, owing to the elevation of LCST of the polymer, the immunoreaction could be carried out at a temperature closer to the physiological environment of organism and the rate of immunoreaction was faster. A sandwich fluorescence immunoassay for rabbit IgG and a competitive enzyme-linked fluorescence immunoassay for human IgG were established with satisfactory results. In chapter 3, the superiority of pH-sensitive polymer to temperature-sensitive polymer as immobile phase of FIA was firstly discussed. Poly(N-isopropylacrylamide-methacrylic acid) [P(NIP-MAA)J, a linear water-soluble pH-sensitive polymer whose phase transition temperature is about 5.6 at 37℃, was synthesized and used as a novel separation carrier for the reactants in immunoassay. A fluorescence immunoassay for rabbit IgG was established. Immune reaction time was decreased to 4 min at 37℃. The pH-sensitive mechanism was simply clarified. The immobilization efficiency and non-specific adsorption of antibody on P(NIP-MAA) polymer were evaluated. If antigen or antibody was immobilized by the way of heat initiation instead of oxidation-reduction initiation, the immobilization efficiency, immunological reaction activities of biological active matter and phase transition pH range were improved. Moreover, some environment-sensitive polymer synthesized only in organic solvent or under high temperature could be used as an immunoreaction carrier by heat initiation immobilization. Two immobilization modes were compared in the determination of human IgG. As a little hydrophobic monomer butyl methacrylate (BMA) was introduced to co-polymerized with INP and MAA in organic solvent, the lower critical solution pH (LCSP) was increased to 6.0, which was more suitable to FIA. Then a competitive FIA for rabbit IgG was established using this pH-sensitive polymer.In chapter 4, Poly(N-isopropylacrylamide-N,N’-dimethyl aminopropyl methacrylamide) [P(NIP-DMAPM)], a linear novel water-soluble pH-sensitive polymer whose phase transition temperature is about 7.4 at 37℃, was synthesized. The P(NIP-DMAPM) precipitated out of water above a critical pH 7.4 at 37℃ and redissolved at pH below 7.4. P(NIP-DMAPM) was used as a novel separation carrier for the reactants in immunoassay and a fluorescence immunoassay for rabbit IgG and human IgG was built. Because of neutral LCSP, the damage to antigen-antibody immune complexation was greatly decreased in the course of separation. The nonspecific adsorption and inhibition of更多还原