CN103995122A - 钯-金合金纳米笼免疫层析试纸条检测微囊藻毒素-lr的方法 - Google Patents
钯-金合金纳米笼免疫层析试纸条检测微囊藻毒素-lr的方法 Download PDFInfo
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Abstract
钯-金合金纳米笼免疫层析试纸条检测微囊藻毒素-LR的方法，属免疫分析化学技术领域。以Pd纳米颗粒为晶种子制备出具有中空结构的Pd-Au双金属纳米笼，用于标记微囊藻毒素-LR单克隆抗体喷涂到结合垫，于检测线上喷涂BSA偶联微囊藻毒素-LR，以羊抗鼠IgG喷涂控制线制得用于快速检测水体中微囊藻毒素-LR的免疫层析片条，用竞争免疫层析法，通过读取检测线上滞留的金纳米笼-微囊藻毒素抗体结合物的灰度值，对试样中微囊藻毒素-LR进行定量分析。由于合金中两种金属元素的协同作用，Pd-Au合金对蛋白质分子中的巯基和氨基结合力高于单金属金纳米笼的结合力，因此在用Pd-Au合金纳米笼标记微囊藻毒素抗体时，比用金纳米笼标记时更容易，形成的Pd-Au合金纳米笼-微囊藻毒素抗体结合物更稳定。
Description
技术领域
本发明属免疫分析化学技术领域。
背景技术
富营养化严重的淡水湖泊，每年夏天气温上升均要引起蓝藻爆发，造成严重污染。在蓝藻大家族中，有一些种类会分泌较强的毒素[见史红星,曲久辉,王爱民等,官厅水库蓝绿藻及藻毒素初步研究，高等学校化学学报,2005,26(9):1650-1652]，而微囊藻毒素(microcystins，MCs)是其中的典型代表。其中2，4位上含亮氨酸和精氨酸的MCs-LR是已知的最易出现和毒性最强的环状七肽肝毒素，是淡水藻类植物中的促癌因子[见Yu S.Z.,Chen G.,Zhi X.L.,et al.Primary liver cancer:natural toxins and prevention in China(J).Toxicol Sci,1998,23(Suppl2):143-148；以及见俞顺章，赵宁,资晓林等，饮水中微囊藻毒素与我国原发性肝癌关系的研究，(J).中华肿瘤杂志,2001,23(2):96-99]，具有肾毒性、免疫毒性和明显的肝毒性，动物饮用藻类毒素污染的水可发生中毒甚至死亡[见Carmichael W.W.,Jones C.L.A.,Mahmood N.A.,et al.Algal toxins and water baseddisease(J).CRC Crit,Rev.Environ.Control1985；15:275-313]。同时，微囊藻毒素是蛋白磷酸酶的抑制剂，它能够激活人体内的癌基因，同时抑制抗癌基因，使抗癌基因失活，使癌症发生的可能性提高近10倍。有研究表明，长期通过喝水接触此类毒素可能会诱发原发性肝癌,提高肿瘤的活性引发死亡[见陈刚,俞顺章,卫国荣等.肝癌高发区饮用水型中微囊藻毒素含量调查(J)中华预防医学杂志，1996；30(1):6-9；以及见Gupta N.,Pant S.C.,Vijayaraghavan R.,et al,Comparative toxicity evaluation of cyanobacterial cyclic peptide toxin microcystinvariants(LR,RR,YR)in mice(J),Toxicology,2003,188(2-3):285–296.以及Puerto M,Pichardo S,Jos A,et al,Comparison of the toxicity induced bymicrocystin-RR and microcystin-YR in differentiated and undifferentiated Caco-2cells(J),Toxicon,2009,54(2):161-169.]。而自来水厂的氯消毒过程不仅不能除去藻类毒素，反而有可能增加其致突活性[见崔莹,徐祖信,尹海龙.水中藻类污染物及其藻毒素分析方法研究进展(J).安徽农业科学.2008，36(29)：12873-12875,12878]。WHO和我国限定饮用水中微囊藻毒素MC-LR含量不得大于1.0μg/mL(GB5749-2006)。因此，建立灵敏、特异的水中微囊藻毒素检测方法具有重要的意义。
微囊藻毒素-LR(Microcystin-LR)，分子式为C49H74N10O12，分子量为995D(道尔顿)，其结构式见图1。
微囊藻毒素的检测方法可分为生物测试法、化学分析法、生化分析法等。；化学检测包括毛细管电泳(CE)、核磁共振(NMR)、色质谱联用(LC-MS)等,其中使用最为广泛和成熟的方法是高效液相色谱(HPLC)。生化分析法包括酶联免疫吸附法和蛋白磷酸酶抑制法，如Camp`as等采用竞争酶联免疫法和丝网印刷电极实现了对微囊藻毒素的安培检测。鞠熀先等将抗体共价结合到富含羧基的碳纳米角上构建了灵敏的竞争型微囊藻毒素电化学免疫传感器。以上方法不仅依赖大型仪器设备，而且需要具有专门操作技术人员,不能用于现场分析,限制了这些方法的普及应用。
近几年随着生化免疫技术以及酶联免疫技术的进一步发展，快速、简便、易操作的免疫层析技术法开始被广泛地应用到水体中微囊藻素的检测中。但所用标记物金溶胶存在容易团聚，不易保存等缺点。
本专利申请人在此以前申请号为201310610348.8的中国专利申请，公开了一种免疫层析试纸条检测微囊藻毒素-LR的方法，通过一步制备法制得具有中空结构的金纳米笼(Au NCs)，将其标记到Anti MC-LR上制得金纳米笼-微囊藻毒素抗体结合物，将BSA偶联的微囊藻毒素-LR、IgG分别喷涂于硝酸纤维素膜上作检测线以及控制线制得免疫层析片条；利用竞争免疫层析法，通过读取检测线上滞留的金纳米笼-微囊藻毒素抗体结合物的灰度值，对试样中微囊藻毒素-LR进行定量分析。不但克服了试纸条技术中金溶胶不易保存的缺点，而且操作简单，抗体的修饰需要的时间较短。由于技术在不断进步，还需要在此基础上进一步开发出更好的免疫层析试纸条检测微囊藻毒素-LR的方法。
合金纳米材料又称二元或多元金属纳米材料，是由两种或两种以上的金属或非金属所组成的具有金属特性的物质。由于合金中两种金属元素的协同作用而导致合金的催化活性大大增加并且具有许多独特的性能，近年来引起了国内外众多研究者的兴趣。Zhang等首先利用水热法合成了平均由147个Pd原子组成的Pd纳米颗粒，然后在N2的保护下将Au原子从溶液中置换到Pd纳米颗粒上，成功合成的Pd-Au双金属纳米颗粒，它的化学催化性能和生物相容性比单一的Pd、Au金属纳米颗粒的更加令人满意[Zhang H,Watanabe T,Okumura M,et al.Catalytically highly active top gold atom on palladium nanocluster[J].Naturematerials,2012,11(1):49-52.]。Xia等通过相继将HAuCl4和Na2PdCl4加到Ag纳米立方体中还原得到Ag/Au/Pd三金属纳米笼[Cobley,C.M.；Campbell,D.J.；Xia,Y.Tailoring the optical and catalytic properties of gold-silver nanoboxes andnanocages by introducing palladium.Adv.Mater.2008,20(4):748-752]。他们还通过先合成Pd纳米立方体，以此为晶种子，再加入HAuCl4用抗坏血酸在Pd晶种子上还原出金制得Pd-Au合金纳米立方体[Lim B.K.,Li Z.Y.,Xia Y.,et.al.Synthesis ofPd-Au bimetallic nanocrystals via controlled overgrowth[J].J.Am.Chem.Soc.2010,132(8):2506-2507.]。但到目前为止，未见有将Pd-Au合金纳米颗粒应用于免疫层析技术的报道。
发明内容
本发明的目的是克服目前常用着色标记物金纳米颗粒溶胶不宜保存的缺点，提供相比申请号为201310610348.8的中国专利申请技术性能更好的方法，即一种钯-金合金纳米笼免疫层析试纸条检测微囊藻毒素-LR的方法。
本发明方法如下：首先以Pd纳米颗粒为晶种子制备出具有中空结构的Pd-Au双金属纳米笼，并用于标记微囊藻毒素-LR单克隆抗体喷涂到结合垫，于检测线上喷涂BSA偶联微囊藻毒素-LR，以羊抗鼠IgG喷涂控制线制得用于快速检测水体中微囊藻毒素-LR的免疫层析片条，用竞争免疫层析法，通过读取检测线上滞留的金纳米笼-微囊藻毒素抗体结合物的灰度值，对试样中微囊藻毒素-LR进行定量分析。
所说以Pd纳米颗粒为晶种子制备具有中空结构的Pd-Au双金属纳米笼，其方法虽然为现有技术，但最好按如下过程进行：将54～56mL PVP，298～302mgL-抗坏血酸、1498～1502mg KBr和284～286mg氯钯酸混合并加热至沸腾，在匀速搅拌下反应2.9～3.5小时，冷却到室温后，离心分离产物，并用无水乙醇和去离子水反复冲洗三到五次，以除去其中溶解的溴化物，所得产物密封保存做下一步合成的种子；用以上一步合成的Pd纳米笼作为晶种子合成Pd-Au纳米笼颗粒，取19～21ml的去离子水，加入0.48～0.51ml已浓缩好的Pd纳米立方体溶液，4.9～5.1mgPVP和2.9～3.1mgL-1抗坏血酸，将此混合液加热到沸腾反应19～21min，并保持匀速搅拌，将10-12mg氯金酸分散到1.9～2.1ml的去离子水中，并逐滴加入到上述反应液中，再将该混合液加热至沸腾状态反应14～16min，待温度冷却到室温，离心分离产物，用无水乙醇和去离子水洗涤，浓缩即得。
考察了Pd-Au NCs/Anti MC-LR结合物的用量以及检测底液等条件对检测结果的影响。在最优条件下，片条对微囊藻毒素-LR浓度响应范围为0.1ng/mL至50ng/mL，检测下限为0.03ng/mL。
该方法操作简单，无需大型仪器或专业技术人员，可用于微囊藻毒素的现场快速检测。
本发明方法与申请号为201310610348.8的中国专利申请技术使用性能对比如下：Pd-Au双金属纳米笼状颗粒的光吸收截面高于金纳米笼的吸收截面,因此基于Pd-Au双金属纳米笼状颗粒制备的免疫层析片条检测微囊藻毒素的灵敏度比申请号为201310610348.8的中国专利申请技术的灵敏度高，响应信号明显增强。图2为不同标记物制作的免疫层析片条对相同浓度微囊藻毒素的灰度响应值比较，从图中可看出，Pd-Au双金属纳米笼为标记物的片条的响应明显大于用金纳米笼为标记物的片条的响应。
本发明的有益效果：和申请号为201310610348.8的中国专利申请技术相比，本发明的优点是：由于合金中两种金属元素的协同作用，Pd-Au合金对蛋白质分子中的巯基和氨基结合力高于单金属金纳米笼的结合力，因此在用Pd-Au合金纳米笼标记微囊藻毒素抗体时，比用金纳米笼标记时更容易，形成的Pd-Au合金纳米笼-微囊藻毒素抗体结合物更稳定。
附图说明
图1为微囊藻毒素结构式。
图2不同标记物的免疫层析片条对同浓度微囊藻毒素的响应灰度值比较
图3为实施例Pd-Au纳米笼的透射电镜图。
图4为免疫层析片条结构示意图。
图5为实施例不同浓度的MC-LR的测试图片。
图6为实施例免疫层析片条的校准曲线。
图7为不同的展开底液对检测结果的影响
图8为检测线喷涂次数对检测结果的影响
图9为Pd-Au纳米笼标记单克隆抗体用量对检测结果的影响。
具体实施方式
实施例，其步骤为：
1钯-金合金纳米笼Pd-Au NCs的制备
钯-金合金纳米笼按下述方法制备：参照修改过的Xia等的方法制备金纳米笼[Lim B.K.,Li Z.Y.,Xia Y.,et.al.Synthesis of Pd-Au bimetallic nanocrystals via controlledovergrowth(J).J.Am.Chem.Soc.2010,132(8):2506-2507.]。将55mL PVP(MW＝38000)，300mgL-抗坏血酸、1500mg KBr和285mg氯钯酸混合并加热到80℃(至沸腾)，在匀速搅拌下反应3小时，冷却到室温后，离心分离产物，并用无水乙醇和去离子水反复冲洗三到五次，以除去其中溶解的溴化物。所得产物密封保存做下一步合成的种子。
以上一步合成的Pd纳米笼作为晶种子合成Pd-Au纳米笼颗粒。取20ml的去离子水，加入0.5ml已浓缩好的Pd纳米立方体溶液,5mgPVP和3mgL-1抗坏血酸。将此混合液加热到沸腾反应20min，并保持匀速搅拌。将10-12mg的氯金酸分散到2ml的去离子水中，并逐滴加入到上述反应液中，再将该混合液加热至沸腾状态反应15min。待温度冷却到室温，离心分离产物，用无水乙醇和去离子水洗涤，浓缩5倍，密封保存以后待用。图3为制得的Pd-Au纳米笼的透射电镜图。
2钯-金合金纳米笼标记单克隆抗体结合物的制备及纯化
通过参照Peng Zhaofeng等[Gupta N.,Pant S.C.,Vijayaraghavan R.,et al,Comparativetoxicity evaluation of cyanobacterial cyclic peptide toxin microcystin variants(LR,RR,YR)inmice(J),Toxicology,2003,188(2-3):285–296.]的方法合成该结合物。将所合成的钯-金合金纳米笼用于标记单克隆抗体制得结合物(Pd-Au NCs-Anti MC-LR)。取125μL经五倍浓缩过的Pd-Au NCs溶液，用200mM碳酸钠调节pH为9。加入1.2mg/mL Anti MC-LR抗体5μL，每次加1μL，间隔三分钟，滴加完成后，室温振摇2h。为封闭Pd-Au NCs的多余的结合位点，将12.5μL BSA(10％)滴加进混合溶液中继续振摇0.5h。随后以14000r/min转速，低温(12℃)离心15min，弃去上层清液，下层物质用PBS洗涤两次，最后分散在0.5mL洗脱液(20mmol/LNa3PO4·12H2O,5％BSA,0.25％Tween20,和10％蔗糖)中。获得的Pd-AuNCs/Anti MC-LR溶液4℃保存备用。
3免疫层析试纸条的制备
样品垫GF-08裁剪成16mm×30cm长条，浸泡于预先配制好的样品垫处理液中(0.25％Triton X-100,0.05mol/L Tris–HCl,和0.15mmol/L NaCl)30min后烘干备用。将玻璃纤维的金胶结合垫裁剪成0.8～1.0cm×30cm长条，同时将吸收垫裁剪成1.7cm×30cm长条备用。
选取不同的硝酸纤维素膜Millipore135或Millipore180在喷条机BiodotXYZ3060上进行喷片操作。将二抗(羊抗鼠IgG)稀释成2mg/mL后用喷片机喷涂在距硝酸纤维素膜边缘1cm处做为控制线(C线)，然后将Microcystins-BSA稀释成0.2mg/mL喷涂在距硝酸纤维素膜另一边1cm处作为检测线(T线)，喷涂不同次数时,每次都要在37℃烘干后再喷涂。最后喷涂完成后于37℃烘干2h。片条按图2所示组装，其中1为样品垫，2为硝酸纤维素膜，3为PVC底板，4为吸收垫，5为检测线(T线)，6为控制线(C线)，7为结合物释放垫，箭头表示流向(见图4)。
4样品的测定
测试之前将3μL Pd-Au NCs-Anti MC-LR滴在金胶结合垫上并干燥10min。将微囊藻毒素-LR用0.01mol/L磷酸盐缓冲溶液(PBS)配制成不同浓度的标准溶液，各取80μL，将片条放入溶液中展开10min。溶液中的微囊藻毒素-LR抗原与一部分Pd-Au纳米笼/抗体结合物(Pd-Au NCs/Anti MC-LR)结合形成Pd-AuNCs/Anti MC-LR/MC-LR免疫复合物，由于毛细管作用带动着未结合抗原的Pd-Au纳米笼/抗体结合物(Pd-Au NCs/Anti MC-LR)一起流动到T线上，此时T线上固定的BSA-MC-LR与溶液中的MC-LR竞争结合Pd-Au NCs/AntiMC-LR，将还未结合抗原的Pd-Au NCs/Anti MC-LR捕获在T线上形成Pd-AuNCs/Anti MC-LR/BSA-MC-LR免疫结合物,并在检测线上聚积显现出一条可见的色带。Pd-Au NCs-Anti MC-LR结合溶液中抗原(MC-LR)所形成的Pd-AuNCs/Anti MC-LR/MC-LR免疫复合物继续流动，到固定有二抗(羊抗鼠IgG)的C线上被捕获。此时，再加入30μL PBS冲洗片条。将片条放入读条机中，即可用读取片条上T/C线的灰度值。测试溶液中微囊藻毒素-LR的含量越高，竞争结合Pd-Au NCs-Anti MC-LR的可能性越大，则T线上结合Pd-Au NCs-Anti MC-LR的可能性就越低，颜色越浅。与此相反，固定有二抗(羊抗鼠IgG)的C线捕获免疫复合物(Pd-Au NCs/Anti MC-LR)的可能性越大，颜色越深。根据不同标准浓度的溶液对应着不同灰度值，即可获得检测微囊藻藻毒素-LR的标准校正曲线，进而实现对未知水样中的微囊藻毒素-LR进行定量分析。
图5为不同浓度的MC-LR的测试图片。图6为免疫层析片条的校准曲线。
优化条件的实验
测试底液对检测结果影响
考察了PBS、PBS+BSA、PBS+Tween、PBS+BSA+Tween四种不同底液对片条检测结果的影响，结果见图7。从中可以看出，PBS做检测底液时检测效果最好，所以选取PBS为测试底液。
检测线喷涂次数对检测结果的影响
由于吸附在硝酸纤维素膜上的BSA-MC-LR总量受喷涂次数的影，进而对灰度值的检测也有很大的影响。因此，实验选取同一批次制作的、且检测线分别喷涂1次、2次、3次的片条对同一浓度的微囊藻毒素-LR溶液进行测量，结果如图8所示。由图8可以看出，喷涂次数为2次时灰度值最大，检测结果最好，故选取T线喷涂两次为最后条件。
Pd-Au纳米笼标记单克隆抗体用量的影响
也考察了Pd-Au纳米笼标记单克隆抗体(Pd-Au NCs/Anti MC-LR结合物)的不同滴加量对同一浓度标准溶液检测结果产生的影响。结果见图9。由图9可知，在金纳米笼标记单克隆抗体量为3μL时，检测灰度值已经很大，为节约用量，固定每次测试时滴加的金纳米笼标记单克隆抗体量为3μL。
硝酸纤维素膜的选择
硝酸纤维素膜的不同，会影响检测结果的灵敏度。实验选取了HF135和HF180两种不同硝酸纤维素膜在相同的实验条件下制作层析片条并对同一浓度的标准溶液进行了对比检测实验，结果见表1。由表1可知使用HF135的硝酸纤维素膜的片条检测效果优于使用HF180的硝酸纤维素膜的片条，相对标准偏差仅为4.77％。因此选取HF135型号的硝酸纤维膜制作片条。
表1两种不同的硝酸纤维素膜检测结果对比
Claims (3)
1.一种钯-金合金纳米笼免疫层析试纸条检测微囊藻毒素-LR的方法，其特征在于：首先以Pd纳米颗粒为晶种子制备出具有中空结构的Pd-Au双金属纳米笼，并用于标记微囊藻毒素-LR单克隆抗体喷涂到结合垫，于检测线上喷涂BSA偶联微囊藻毒素-LR，以羊抗鼠IgG喷涂控制线制得用于快速检测水体中微囊藻毒素-LR的免疫层析片条，用竞争免疫层析法，通过读取检测线上滞留的金纳米笼-微囊藻毒素抗体结合物的灰度值，对试样中微囊藻毒素-LR进行定量分析。
2.如权利要求1所述的钯-金合金纳米笼免疫层析试纸条检测微囊藻毒素-LR的方法，其特征在于：将54～56mL PVP，298～302mgL-抗坏血酸、1498～1502mg KBr和284～286mg氯钯酸混合并加热至沸腾，在匀速搅拌下反应2.9～3.5小时，冷却到室温后，离心分离产物，并用无水乙醇和去离子水反复冲洗三到五次，以除去其中溶解的溴化物，所得产物密封保存做下一步合成的种子；用以上一步合成的Pd纳米笼作为晶种子合成Pd-Au纳米笼颗粒，取19～21ml的去离子水，加入0.48～0.51ml已浓缩好的Pd纳米立方体溶液，4.9～5.1mgPVP和2.9～3.1mgL-1抗坏血酸，将此混合液加热到沸腾反应19～21min，并保持匀速搅拌，将10-12mg氯金酸分散到1.9～2.1ml的去离子水中，并逐滴加入到上述反应液中，再将该混合液加热至沸腾状态反应14～16min，待温度冷却到室温，离心分离产物，用无水乙醇和去离子水洗涤，浓缩即得。
3.如权利要求2所述的钯-金合金纳米笼免疫层析试纸条检测微囊藻毒素-LR的方法，其特征在于：以PBS为测试底液，T线喷涂两次，每次测试时滴加的金纳米笼标记单克隆抗体量为3μL，以HF135型号的硝酸纤维膜制作片条。
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| US20070121113A1 (en) * | 2004-12-22 | 2007-05-31 | Cohen David S | Transmission-based optical detection systems |
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| YOGESH NANGIA等: "Palladium@gold bimetallic nanostructures as peroxidase mimic for development of sensitive fluoroimmunoassay", 《ANALYTICA CHIMICA ACTA》 * |
| 郑丽等: "基于银钯铂复合纳米材料标记的癌胚抗原免疫传感器的研制", 《云南大学学报(自然科学版)》 * |
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