Abstract
去除荧光标记后残余荧光染料可以提高荧光颗粒检测的灵敏度、准确度和效率。该文发展了一种原位电泳洗脱(electrophoretic elution, EE)模型,用于在荧光标记后快速去除多余的荧光探针,实现荧光颗粒的灵敏检测。将牛血清蛋白(BSA)和磁珠(MBs)作为模式蛋白和微颗粒,混合孵育获得MBs-BSA,用异硫氰酸荧光素(FITC)对MBs-BSA标记,得到MBs-BSAFITC复合物。将含有多余FITC的MBs-BSAFITC溶液与低凝聚温度琼脂糖凝胶溶液按1:5的体积比混合,并将混合物凝胶和纯琼脂糖凝胶分段填充到电泳通道中。电泳过程中,利用颗粒尺寸与凝胶孔径的差异来保留MBs-BSAFITC,同时将游离的FITC洗脱。经过30 min的电泳洗脱,通道内多余的FITC清除率达到97.6%,同时目标颗粒荧光信号保留了27.8%。成像系统曝光时间为1.35 s时,电泳洗脱将颗粒与背景的荧光信号比(P/B ratio, PBr)从1.08增加到12.2。CCD相机的曝光时间增加到2.35 s,可以将PBr提高到15.5,可进一步实现对微弱荧光亮点的高灵敏检测。该模型有以下优点:(1)能对颗粒表面非特异性吸附的FITC实现有效洗脱,提高了检测的特异性;(2)能够将97%以上的游离FITC清除;(3) 30 min内能够使凝胶内的背景荧光大幅降低,提高了PBr和检测灵敏度。因此,该方法具有在凝胶中进行基于磁珠/荧光颗粒点的免疫检测、在免疫电泳或凝胶电泳中对蛋白质/核酸条带进行荧光染色等领域的应用潜力。
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