| 应用电致化学发光分子探针技术对微小原甲藻的检测 |
| 摘要点击 1607 全文点击 788 修订日期：2011-08-01 |
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| 中文关键词 电致化学发光 双特异分子探针 微小原甲藻 联吡啶钌 三丙胺 |
| 英文关键词 electrochemiluminescence (ECL) sandwich hybridization integrated with nuclease protection assay (NPA-SH) Prorocentrum minimum (Pavillard) Schiller Ru(bpy)3Cl2·6H2O tripropylamine (TPrA) |
| 作者 | 单位 | E-mail | | 朱霞 | 中国海洋大学海洋生命学院, 青岛 266003 | myfavorite_1314@163.com | | 甄毓 | 中国海洋大学环境科学与工程学院, 海洋环境与生态教育部重点实验室, 青岛 266100 | zhenyu@ouc.edu.cn | | 米铁柱 | 中国海洋大学环境科学与工程学院, 海洋环境与生态教育部重点实验室, 青岛 266100 | | | 于志刚 | 中国海洋大学化学化工学院, 海洋化学理论与工程技术教育部重点实验室, 青岛266100 | | | 池振明 | 中国海洋大学海洋生命学院, 青岛 266003 | | | 路兴岚 | 中国海洋大学环境科学与工程学院, 海洋环境与生态教育部重点实验室, 青岛 266100 | |
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| 中文摘要 |
| 以双特异分子探针技术(NPA-SH)为基础,运用电致化学发光技术(ECL)和磁性微球分选技术对其进行改进,成功建立了用于赤潮藻类定性定量分析的电致化学发光分子探针(ECL-MP)检测新技术.设计微小原甲藻特异性NPA探针并对其进行联吡啶钌和生物素标记,优化磁性微球使用量,在ECL检测装置中启动电化学反应,建立光信号与微小原甲藻细胞数目间的ECL-MP分析曲线并对其进行验证.结果表明,标记后的NPA探针具有一定的特异性和实用性; 4 μg是检测20 μL目标藻杂交混合液的最适磁性微球使用量; 在最适条件下,微小原甲藻细胞数的线性分析范围6.25×102~4×104个; 比较ECL-MP和显微计数方法的样品检测结果,在95%置信区间内二者无显著差异(t-检验).ECL-MP方法为实现微小原甲藻现场样品的快速准确检测提供了一种新方法. |
| 英文摘要 |
| In this study, the electrochemiluminescence-molecular probe (ECL-MP) was established based on the sandwich hybridization integrated with nuclease protection assay (NPA-SH) improved by electrochemiluminescence (ECL). It can be used for detecting Prorocentrum minimum (Pavillard) Schiller qualitatively and quantitatively. NPA probes of P. minimum were designed based on the NPA-SH. After labeled with Ru(bpy)2+3and biotin, they bounded with streptavidin-coupled magnetic beads and generated electrochemiluminescence in an ECL analyzer. The ECL counts per second (CPS) was drawn against the cell number obtained from microscopy to establish a calibration curve of P. minimum. The results showed that the labeled NPA probes had good specificity and practicability, the optimal usage of magnetic beads was 4 μg for detecting 20 μL hybridization mixture, the detection range of P. minimum cell numbers was 6.25×102-4×104, and there was no significant difference between the data gained from ECL-MP and microscopy with 95% confidence level (t-test) when treated with cultured and mixed samples. ECL-MP was a convenient new tool for rapid assessment of P. minimum in marine environment. |
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