中文摘要：

该文发展了一种基于表面连接酶反应及生物金属化的电化学方法用于单碱基突变的检测。金电极表面先固定捕获探针，然后和待检测的突变链及5’端磷酸化的连接探针杂交，在连接酶存在下，表面固定的探针会和连接探针连接起来。如果表面固定的探针和目标链不匹配，则连接反应不能进行。在90℃下进行热变性处理，形成的双链解开，进行了连接反应的连接探针连接到了金电极上。接着，生物素化的检测探针和连接产物杂交，再和链亲和素化的碱性磷酸酶反应，在碱性磷酸酶的作用下，非还原性的抗坏血酸磷酸酯（AA-P）转化成还原性的抗坏血酸（AA）。溶液中的银离子在抗坏血酸的作用下，被还原生成银，从而使得银颗粒沉积在电极表面。用线性扫描伏安法来检测电极表面沉积的银。该方法已成功的用于检测编码K-Ras癌基因的第12位密码子的单碱基突变，检测下限低达80fmol／L。

英文摘要：

A highly sensitive electrochemical method for point mutation detection that utilizes surface enzymatic ligation reaction and biometallization is demonstrated. In this method the surface - immobilized allele - specific probe, complementary to the mutant target, undergoes allele - specific ligation with the 5 ＇ - phosphorylated ligation probe in the presence of the mutant oligonucleotide target and E. coli DNA ligase. If there is an allele mismatch, no ligation takes place. After thermal treatment at 90℃, the formed duplex melts apart, which merely allows the ligation product to remain on the electrode surface. Then, biotinylated detection probes hybridize with the ligation product. With the binding of streptavidin - alkaline phosphatase （SA - ALP） to the biotinylated probes, a non - reductive substrate of alkaline phosphatase, ascorbic acid 2 - phosphate （AA - P）, can be converted into ascorbic acid （AA） at the electrode surface. Silver ion in solution are then reduces by AA, resulting in the deposition of silver metal onto the electrode surface. Linear sweep vohammetry （LSV） is used to detect the amount of deposited silver. The proposed approach has been successfully implemented for the identification of single base mutation in codon 12 of K - ras oncogene target with a detection limit of 80 fmol/L, demonstrating that this method provides a highly specific, sensitive and cost - efficient approach for point mutation detection.