Facile preparation of streptavidin-coated sephadex beads and their application to chemiluminescence detection of a target DNA
Facile preparation of streptavidin-coated sephadex beads and their application to chemiluminescence detection of a target DNA
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链霉亲和素包被的葡聚糖凝胶珠的简便制备及其在目标 DNA 化学发光检测中的应用
DOI：
10.1007/s00604-014-1348-9
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Kai M
中科院分区：
文献类型：
--
作者：
El-Mahdy AF;Ejupi V;Shibata T;Kabashima T;Lu J;Kai M
报道了一种制备链霉亲和素(SA)包衣葡聚糖珠的有效方法，即通过将氧化葡聚糖链亲和素(SA)上的游离氨基与氧化葡聚糖珠上的醛还原胺化来制备SA包衣葡聚糖珠。所得微球具有稳定的球形，平均直径为20-100nμm。每1 mg微球的SA偶联容量分别是市售SA包覆聚苯乙烯和琼脂糖球的~24倍和~5倍。新型微球通过夹心型杂交方法成功地应用于端粒DNA的化学发光检测，该方法包括以下步骤：(A)首先将目标DNA与固定在SA包被的葡聚糖凝胶小球上的生物素化捕获c DNA杂交；(B)然后将杂交体与富含鸟嘌呤的聚合物探针夹心；(C)在室温下与3，4，5-三甲氧基苯乙二醛试剂混合10 h后，用化学发光法灵敏地检测到端粒DNA。整个分析过程可在2-3小时内完成，目标DNA的浓度在5-200 NM范围内呈线性关系。在试管中，检测下限约为0.75nM(1.3ng · 100ngμL−1)。与其他方法相比，该方法具有线性范围宽、检测下限低、速度快等优点。我们推测，这种新的方法和材料将很容易地应用于对目标DNA的灵敏检测，也适用于基于微球的化学发光免疫分析。将制备的微球应用于夹心型DNA杂交法对目标DNA的化学发光检测。该方法对含有端粒序列的靶DNA具有较高的敏感性和较好的特异性。
We report on an efficient strategy for the preparation of streptavidin (SA)-coated sephadex beads by the reductive amination of the aldehyde groups on the oxidized sephadex beads that were coupled to the free amino groups of SA. The resulting beads have a stable spherical shape with an average diameter of 20–100 μm. The capacity of SA conjugated to 1 mg of these beads is, respectively, ~24 and ~5 times higher than that of commercially available SA-coated polystyrene and sepharose beads. The new beads were successfully applied to the chemiluminescence (CL) detection of telomer DNA via a sandwich-type hybridization assay that involves the following steps: (a) Target DNA is first hybridized with biotinylated capture cDNA immobilized on the SA-coated sephadex beads; (b) the hybrid is then sandwiched with a guanine-rich polymeric probe which (c), is sensitively detected by CL after mixing with a 3,4,5-trimethoxy-phenylglyoxal reagent at room temperature for 10 s. The entire assay can be completed within 2–3 h and has a linear range that extends from 5 to 200 nM concentrations of the target DNA. The lower detection limit is approximately 0.75 nM (1.3 ng · 100 μL−1) in a test tube. Compared to other methods, this one has a wider linear range, a lower detection limit, and is faster. We presume that the new method and materials will be readily applicable to the sensitive detection of target DNA, and also in bead-based CL immunoassays.Graphical AbstractWe describe a facile strategy for the preparation of streptavidin (SA)-coated sephadex beads with high capacity of SA. The prepared beads were applied to the chemiluminescence detection of a target DNA via a sandwich-type DNA hybridization assay. This assay exhibited high sensitivity and good specificity for the target DNA having a telomere sequence.