新型pH敏感相分离高分子的制备及其在免疫分析中的应用

聚Ｎ 异丙基丙烯酰胺 (ＰＮＩＰ)温度敏感高分子以其独特的温度敏感性质已成功地应用于免疫分析[1～ 6] .但这类温度敏感相分离免疫分析的反应温度必须控制在ＰＮＩＰ的相转变温度 ( 31℃ )以下 ,这不可避免地会影响免疫反应速率 .ｐＨ敏感高分子是另一类对环境敏感的智能型高分子 ,在其相转变ｐＨ值附近发生沉淀与溶解的可逆性变化 .目前 ,ｐＨ敏感高分子在免疫分析中的应用并没有受到重视 ,研究得较少[7] .这主要是由于 ｐＨ敏感高分子的相转变 ｐＨ值大都在 3左右 ,在此ｐＨ条件下 ,免疫反应生成的抗原 抗体免疫复合物会受到不同程度的…     

两种环境敏感高分子在相分离免疫分析中的比较研究

进行了两种环境敏感高分子在相分离免疫分析嗜水气单胞菌外膜蛋白(outer membrane protein, OMP)中的比较研究. 首先合成温度敏感高分子聚N-异丙基丙烯酰胺和pH敏感高分子聚(N-异丙基丙烯酰胺-甲基丙烯酸), 分别以N-羟基琥珀酰亚胺丙烯酸酯(NAS)和碳二亚胺(EDCI)作为偶联剂与抗OMP抗体(Ab)偶联形成抗体复合物(Ab-polymer), 在竞争型免疫测定中, OMP标准溶液与异硫氰酸荧光素标记OMP在均相条件下竞争性地与Ab-polymer反应, 调节外界环境分离出高分子免疫复合物沉淀, 重新溶解后荧光法定量, 两种体系的OMP浓度均在400～3000 ng/mL范围内与荧光强度呈良好线性关系, 检出限分别为84.7和39.6 ng/mL. pH敏感高分子相比于温度敏感高分子具有以下优点: 可以在37 ℃的生理温度进行免疫反应, 进一步提高了免疫反应的速度和效率; 可利用高分子本身的活性基团进行Ab的固定, 固定化效率、固定Ab的免疫反应活性较之NAS偶联法得到了提高; 有更高的检测灵敏度. 因此, pH敏感高分子更适合于作为相分离免疫分析的载体.     

新型pH敏感高分子相分离荧光免疫分析法测定兔血清中IgG

通过将N-异丙基丙烯酰胺（NIP）、甲基丙烯酸丁酯（BMA）和甲基丙烯酸（MAA）共聚得到了一种新型的pH敏感高分子,其相转变pH（pHtr）在37℃下为6.0;当溶液pH〉6.0时,高分子溶解;pH〈6.0时,高分子很快从溶液中沉淀出来。在竞争型免疫测定中,偶联在高分子上的兔免疫球蛋白G（IgG）和标准兔IgG在溶液中竞争性地与有限量的异硫氰酸荧光素标记抗体反应,根据pHtr以下免疫复合物沉淀的特性进行分离,建立了均相免疫反应、异相分离的兔IgG新型测定方法,线性范围为100—1000μg／L;检出限为10μg/L。方法灵敏、快速且操作简便,由于pHtr较之以前合成的敏感高分子更接近于中性,分离时可降低对抗原-抗体免疫复合物造成的损坏。用于兔血清中免IgG的测量,结果令人满意。     

以萘酰亚胺衍生物基荧光高分子为载体和指示剂的 相分离免疫分析方法

以4-甲氧基-N-(2-N’,N’-二甲基氨基乙基-N’-烯丙基)萘二甲酰亚胺氯化铵(DMNAA)为荧光单体, 合成了一种pH敏感荧光高分子聚N-异丙基丙烯酰胺-4-甲氧基-N-(2-N’,N’-二甲基氨基乙基-N’-烯丙基)萘二甲酰亚胺氯化铵-N,N-二甲基氨丙基甲基丙烯酰胺[P(NIP-DMAPM-DMNAA)]. 采用共聚法将日本血吸虫抗原(SjAg)固定在P(NIP-DMAPM-DMNAA)上, 制备P(NIP-DMAPM-DMNAA)-SjAg连接物, 与日本血吸虫抗体(待测, SjAb)发生免疫反应后, 调节pH值, 使荧光高分子相变分离高分子-免疫组分连接物, 最后, 利用蛋白A对抗体的亲和性捕获P(NIP-DMAPM-DMNAA)-SjAg-SjAb, 通过测定高分子自身的荧光信号来定量 SjAb. 该新型高分子具有良好的荧光特性, 对pH响应快速, 37 ℃下相转变pH值为7.2, 分离免疫复合物时造成的损害低. 与传统相分离免疫分析比较, 新方法通过高分子相变分离和蛋白A捕获双重分离作用, 消除了非特异性组分和未反应的特异性免疫成分等的干扰; 利用高分子自身的荧光信号检测, 无须另外的标记物, 大大提高了免疫分析的简便性. 以日本血吸虫抗体为分析对象, 测得线性范围为1～1500 ng/mL, 抗体检出限为1.3 ng/mL, 相对标准偏差为3.6% (n＝10), 结果令人满意.     

七元瓜环与芦竹碱的主客体相互作用

利用紫外可见吸收光谱法和荧光光谱法分别考察了七元瓜环(Q[7])与芦竹碱(Gramine)在溶液中的主客体相互作用;考察了p H值对主客体相互作用的影响,并利用摩尔比法和Job法计算了主客体相互作用的作用比以及稳定常数;利用等温量热滴定法进一步考察了Q[7]与Gramine主客体相互作用的热力学参数。实验结果表明Gramine与Q[7]在很宽的p H介质条件下(2p H10)都能发生主客体相互作用,在酸性、中性水溶液中形成2∶1的主客体配合物,而在碱性溶液中则形成1∶1的主客体配合物。Gramine与Q[7]的主客体相互作用过程主要是以焓驱动为主的过程。相溶解度实验的结果表明当Q[7]的浓度为1.0×10~(-3)mol·L~(-1)时,可使Gramine的溶解度增大82倍。     

共聚N-异丙基丙烯酰胺单链微凝胶

聚 N -异丙基丙烯酰胺 (PNIPAM)的水溶液具有下临界共溶温度 LCST(约 3 2℃左右 ) ,即当体系温度高于 3 2℃时 ,高分子链的构象发生 Coil- to- Globule的变化 [1～ 3 ] ,而由 N -异丙基丙烯酰胺制备的水凝胶亦存在体积相转变 ,该转变与网络链的构象变化相关 ,文献 [1 ,4,5 ]对从大块凝胶到微米级凝胶的性质进行了研究 .本文成功地制备了聚 N -异丙基丙烯酰胺的单链微凝胶 ,与相应的线性高分子在分子量和化学组成上完全相同 ,初步研究了它们与十二烷基硫酸钠 (SDS)的混合水溶液的粘度性质 ,从而在两者之间建立了直接而明确的联系 .1…     

含氮杂环多芳基咪唑衍生物的合成及其光物理性能

以冰醋酸为溶剂和催化剂,苯偶酰/9,10-菲醌、3-吲哚甲醛/3-咔唑甲醛及醋酸铵经"一锅"反应高效合成了系列含吲哚或咔唑结构单元的多取代咪唑衍生物.考察了反应物配比、溶剂的选择和用量及温度等因素对反应的影响,研究了所合成化合物的光物理性能;筛选出对p H值敏感且结构独特的两个化合物作为p H荧光探针,检测在其作用下MCF-7细胞在不同p H值环境中的荧光成像,结果表明探针2-(9-苄基-9H-咔唑-3-基)-4,5-二苯基-1H-咪唑(2d)和2-(9-苄基-9H-咔唑-3-基)-1H-菲并[9,10-d]咪唑(4)都可用作检测活细胞内p H变化的p H荧光探针.     

一种光响应性热敏聚合物的合成及性能表征

合成了偶氮单体 2 [4 (4′ 乙氧基苯基偶氮 )苯氧基 ]乙基丙烯酸酯 (EAPEA) ,利用核磁共振、傅立叶红外和元素分析法对其分子结构进行了表征 .利用该单体与异丙基丙烯酰胺共聚得到一种对温度和光敏感的共聚物 .共聚物中少量的EAPEA单元能够显著降低聚异丙基丙烯酰胺 (PNIPA)的相转变温度 .当EAPEA的摩尔含量为 2 94%时 ,相转变温度从PNIPA均聚物的 31 8℃下降为 2 2 0℃ .在波长为 36 5nm的紫外光照射下 ,共聚物中的偶氮基团能够从反式构型转变为顺式构型 .在紫外光下照 30s后 ,EAPEA摩尔含量为 0 98%的聚 {异丙基丙烯酰胺 共 2 [4 (4′ 乙氧基苯基偶氮 )苯氧基 ]乙基丙烯酸酯 }的相转变温度从 2 7 2℃上升到2 9 3℃     

新型异核双阳离子型磺酸功能化离子液体的合成及其催化活性

合成了三种基于1-吗啉基-3-咪唑基丙烷异核双阳离子的新型二磺酸功能化BrΦsted酸性离子液体:1-[4-(4-磺酸丁基)吗啉基-3-[3-(4-磺酸丁基)咪唑基]丙烷二(三氟甲烷磺酸)盐{A,[(C4SO3H)m(C4SO3H)i]p(OTf)2}、1-[4-(4-磺酸丁基)吗啉基-3-[3-(4-磺酸丁基)咪唑基]丙烷二硫酸氢盐{B,[(C4SO3H)m(C4SO3H)i]p(HSO4)2}及1-[4-(4-磺酸丁基)吗啉基-3-[3-(4-磺酸丁基)咪唑基]丙烷二磷酸二氢盐{C,[(C4SO3H)m(C4SO3H)i]p(H2PO4)2}.其结构经FT-IR、MS、NMR等确认.选择吗啉、甲基咪唑单磺酸功能化离子液体作为对照组,以苯甲醛与环己酮之间的交叉Aldol缩合反应考察了其催化活性.用Hammett方法测定了其酸性,实验结果表明,所合成的三种双磺酸功能化离子液体的催化能力比单磺酸功能化离子液体的强,其酸性次序与催化活性相一致.其中,阴离子为三氟甲烷磺酸根的离子液体对18种Aldol缩合反应底物有极好的适应性,并且催化活性最强,产率高达90%,在重复使用10次后,仍保持较高的反应活性.关键词二磺酸功能化离子液体;合成;催化活性;Hammett方法;交叉Aldol缩合反应;重复使用     

同时含α-[Mo8O26]4-和β-[Mo8O26]4-的新型超分子化合物的合成、表征及其抗肿瘤活性

以(NH4)2[Mo4O13]·2H2O, NiCl2·6H2O和2-(1H-吡唑-3-基)吡啶为原料, 通过水热方法合成了一个新颖的[Ni(C8N3H7)3]4[Mo8O26]2·7H2O超分子化合物. 采用元素分析、红外光谱和单晶X射线衍射法表征了该无机-有机杂化配合物的结构. 晶体解析表明, 该化合物同时含有α-[Mo8O26]4-和β-[Mo8O26]4-, 在[Ni(C8N3H7)3]2+阳离子和[Mo8O26]4-阴离子之间通过氢键作用结合, 在[Ni(C8N3H7)3]2+阳离子之间通过芳香环的π-π堆积作用连接, 两种[Mo8O26]4-阴离子作为客体共存于一个具有三维蜂窝状的超分子结构中. 同时研究了该化合物对人卵巢癌细胞SK-OV-3、肺癌细胞A549、宫颈癌细胞Hela和乳腺癌细胞MCF-7等细胞的体外抗肿瘤活性. 结果表明, 该化合物对3种不同来源的肿瘤细胞均有一定的增殖抑制作用, 其中对MCF-7细胞的IC50值为6.48 μmol/L, 具有进一步研究的价值.     

基于pH敏感相分离技术的新型荧光免疫测定体系

In this paper, it was discovered that a novel pH-sensitive copolymer of N-isopropylacrylamide (NIP) and N-(3-dimethylaminopropyl)methacrylamide (DMAPM) could be gotten by polymerization. The phase transition pH (pHtr) of P(NIP-DMAPM) polymer was found to be 7.4 at 37℃. The polymer was precipitated out of water above a critical pH=7.4 and re-dissolved below pH----7.4. The characteristic of this polymer made it possible to carry out the immunochemical steps of an immunoassay in a true solution and then to quickly separate the resulting product from the reaction mixture. In a competitive fluorescence immunoassay, the standard rabbit IgG and rabbit IgG immobilized on P(NIP-DMAPM) first competitively reacted with the fluorescein isothiocyanate (FITC) labeled antibody, then the pH of solution was adjusted above the pHtr of polymer to precipitate the polymer-immune complex,and the polymer-immune complex precipitate was separated and re-dissolved by the adjustment of pH, finally the FITC-labeled antibody in the immune complex was quantified by fluorescence measurement. The calibration graph for rabbit IgG was linear over the range of 100-1000 ng/mL with a detection limit of 11 ng/mL. The method is rapid, sensitive and simple. Owing to neutral pHtr of P(NIP-DMAPM), the damage to antigen-antibody immune complex was greatly decreased in the course of separation. In addition, a sandwich enzyme-linked fluorescence immunoassay method for the determination of human IgG was also developed, showing that the pH-sensitive phase separating immunoassay could be performed in the competitive method as well as the sandwich method.     

Comparison of Immobilization Modes in pH-Sensitive Phase Separation Immunoassay

Three immobilization modes of antigen to the polymers in the pH‐sensitive phase separation immunoassay were investigated and compared. The results showed that the immobilization mode in the presence of N‐ethyl‐N′‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDCI) rendered the most desirable results. The immobilization efficiencies and immunological reaction activities of immobilized antigen of this mode were improved over the other two modes. The novel immobilization mode by EDCI was used in the pH‐sensitive phase separation immunoassay for rabbit IgG (Ag). In the competitive immunoassay, immobilized Ag and the standard Ag (or sample) competed for binding to a horseradish peroxidase labeled antibody at 37°C in a homogeneous format. After changing the pH to separate the polymer‐immune complex, the complex precipitate was re‐dissolved and determined by coupling with the color reaction of hydrogen peroxide and o‐phenylenediamine. The linear range of this determination was between 100–1400 ng/mL. Compared to the traditional enzyme‐linked immunosorbent assays (ELISA) using the same reactants, the proposed method was quiet fast (the time decreased from 100?120 to 30 min) and showed similar sensitivity, i.e., 6.0 ng/mL.     

基于可控相转变温度热敏高分子的人IgG酶联荧光免疫分析

以温度敏感高分子聚N－异丙基丙烯酰胺－丙烯酰胺[P(NIP-AA)]作为载体,建立了酶联荧光免疫分析人IgG的新方法。AA摩尔含量为8％的高分子P(NIP-AA)其临界溶解温度为37 °C。竞争型免疫测定中,被固定的IgG和标准溶液（或样品）在33 °C均相条件下竞争性地与辣根过氧化物酶标记抗体反应,升高温度分离出高分子免疫复合物,沉淀重新溶解后通过偶联过氧化氢与对羟基苯乙酸的荧光反应进行定量,线性范围为100-1000 ng/mL,检测限为2.0 ng/mL。方法灵敏、快速操作简便,且提高了免疫反应效率。此外,灵敏度与用传统微孔板做载体相似,但测定时间更快（从100-120分钟减少到30分钟）。该法用于测定人血清中IgG的含量,结果满意。     

带活性末端的热敏高分子的制备及其在荧光免疫分析中的应用

合成了带有活性末端的寡聚N-异丙基丙烯酰胺(ONIPAAm),并考察了其温度敏感性质.以ONI-PAAm作为免疫分析的载体与鼠IgG偶联,以四磺基铁酞菁为标记物,以羊抗鼠IgG抗体为分析模型,建立了竞争型热敏相分离荧光免疫分析新系统.羊抗鼠IgG抗体在0～1500ng/mL范围内与体系相对荧光强度呈良好的线性关系,检测限为2ng/mL.     

Gold nanoparticle-based immunoassay by using non-stripping chemiluminescence detection

A novel microplate-compatible chemiluminescence (CL) immunoassay has been developed for the determination of human immunoglobulin G (IgG) based on the luminol-AgNO(3)-gold nanoparticles CL system. Polystyrene microtiter plates were used for both immunoreactions and CL measurements. The primary antibody, goat-anti-human IgG, was first immobilized on polystyrene microwells. Then the antigen (human IgG) and the gold-labeled second antibody were connected to the microwells successively to form a sandwich-type immunocomplex. The gold label could trigger the reaction between luminol and AgNO(3), accompanied by light emission. Under the optimized conditions, the CL intensity of the system was linear with the logarithm of the concentration of human IgG in the range from 25 to 5000 ng mL(-1), with a detection limit of 12.8 ng mL(-1) ( approximately 80 pM) at a signal to noise ratio of three (S/N = 3). Compared with other reported CL immunoassay method based on gold labels, the proposed CL protocol avoids a strict stripping procedure or difficult to control synthesis processes, making the method more simple, time-saving and easily automated. The present CL method is promising for the determination of clinically important bioactive analytes.     

Determination of proteins at nanogram levels by their quenching effect on large particle scattering of colloidal silver chloride

A novel quantitative method for the determination of proteins in aqueous solutions has been based on the quenching of the resonance scattering light of colloidal silver chloride in the presence of proteins. The detection limits for eight kinds of proteins (BSA, HSA, egg albumin, human gamma-IgG,alpha-chymotrypsin, E. Coli. alpsase, myoglobin, alpha-casein) were at about 8 ng/mL; the linear ranges of the calibration curves were 10-400 ng/mL under optimal conditions,except for human gamma-IgG (20-400 ng/mL), myoglobin (10-300 ng/mL), and alpha-casein (10-300 ng/mL). Three wavelengths (398 nm, 475 nm, 499 nm) were all suitable for the determination and any acidity from pH 3.0 to pH 9.0 could be chosen. A few non-protein substances at high concentration levels interfered with this method, but this problem could simply be overcome by diluting the samples before the assay. Mechanism studies showed that the quenching effect of proteins on the scattering light of colloidal silver chloride was mainly due to the coagulation of AgCl particles retarded by protein. The method was employed for the determination of total protein in human serum with satisfactory results.     

Antibody-biotemplated HgS nanoparticles: extremely sensitive labels for atomic fluorescence spectrometric immunoassay

In this work, antibody goat anti-human IgG as a scaffold was employed for the synthesis and biofunctionalization of HgS nanoparticles (NPs) via a facile one-pot process. After a complete sandwich-type immunoreaction among primary antibody, human IgG and secondary antibody labeled with HgS NPs, a large number of mercury ions released from captured HgS NPs dissolution were quantitatively detected by chemical vapor generation atomic fluorescence spectrometry (CVG-AFS). Taking advantage of the signal amplification property of HgS NPs and the high sensitivity of CVG-AFS, the assay detected human IgG with a limit of detection (S/N = 3) of 0.6 ng mL(-1) (4.0 fmol mL(-1) or 0.4 fmol) and the response was linear over a dynamic range from 1.0 to 5.0 × 10(4) ng mL(-1) with a correlation coefficient of 0.996. A relative standard deviation (RSD) of 1.0 × 10(2) ng mL(-1) human IgG was 1.5% for within-batch (intra-assay) and 4.5% for between-batch (inter-assay). Other proteins, such as goat anti-rabbit IgG, goat anti-human IgG, rabbit anti-human IgG, carcinoembryonic (CEA), α-fetoprotein (AFP), human serum albumin (HSA) and bovine serum albumin (BSA) did not significantly interfere with the assay for human IgG. The analytical result of HgS NPs with AFS-based immunoassay technology for the quantification of human IgG in human serum from patients is in good agreement with the result obtained by conventional immunoturbidimetric method. The consequence shows that the novel immunosensor possessed satisfactory precision, extremely high sensitivity, high selectivity and could be applied for the quantification analysis of real samples.     

基于胶体金标记的阳极溶出伏安免疫分析用于免疫球蛋白G的测定

提出了一种基于胶体金标记的阳极溶出伏安免疫分析方法。免疫反应在聚苯乙烯微孔板中以夹心分析模式进行,通过物理吸附将兔抗人免疫球蛋白G(IgG)抗体固定于微孔板上,与相应抗原IgG发生免疫反应后,再通过夹心模式捕获相应的纳米金标记的羊抗人IgG抗体,然后再与金标羊抗人IgG抗体和金标兔抗羊二抗形成的免疫复合物反应,在微孔板上进一步引入大量的纳米金,将金溶解后,在碳糊电极上用阳极溶出伏安法(ASV)对金离子进行检测,溶出峰电流的大小间接与待分析物IgG的浓度成正比。对免疫分析的一些实验条件进行了优化。阳极溶出峰电流与IgG的对数浓度在1.1~1 143 ng/mL范围内呈良好的线性关系,检出限为1 ng/mL。将该方法应用于人血清中IgG浓度的测定,取得了满意结果。     

雌二醇的水凝胶毛细管电泳免疫分析

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