应用电致化学发光分子探针技术对微小原甲藻的检测

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.

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

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)3(2+) and 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 microg for detecting 20 microL hybridization mixture, the detection range of P. minimum cell numbers was 6.25 x 10(2) - 4 x 10(4), 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.