应用电致化学发光分子探针技术对微小原甲藻的检测  

Detection of Prorocentrum minimum(Pavillard) Schiller with the Electrochemiluminescence-Molecular Probe

作　　者：朱霞[1] 甄毓[2] 米铁柱[2] 于志刚[3] 池振明[1] 路兴岚[2] 

机构地区：[1]中国海洋大学海洋生命学院,青岛266003 [2]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 [3]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

出　　处：《环境科学》2012年第2期640-646,共7页Environmental Science

基　　金：国家自然科学基金项目(40706044,40705047);国家908项目(908-02-02-02)

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;4μg是检测20μL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.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.

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 

分 类 号：X830.2[环境科学与工程—环境工程]