Fojta Miroslav, Havran Ludek, Kizek Rene, Billova Sabina, Palecek Emil
Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, CZ-612 65 Brno, Czech Republic.
Biosens Bioelectron. 2004 Nov 15;20(5):985-94. doi: 10.1016/j.bios.2004.06.015.
In electrochemical DNA hybridization assays target or probe DNAs end-labeled with electroactive compounds have been frequently used. We show that multiple osmium labels yielding faradaic (at carbon or mercury electrodes) and catalytic signals (at mercury electrodes) can be easily covalently bound to DNA molecules. We use (GAA)(7) (T)(n) oligodeoxynucleotides (ODNs) with n ranging between 5 and 50. (T)(n) tails are selectively modified with osmium tetroxide,2,2'-bipyridine leaving the (GAA)(7) repeat intact for the DNA hybridization. These ODNs are applied as reporter probes (RP's) in DNA hybridization double-surface (DS) assay using magnetic beads for the DNA hybridization and pyrolytic graphite (PGE) or hanging mercury drop (HMDE) electrodes for the electrochemical detection. We show that in difference to the usual single-surface methods (where the RP has to be bound to target DNA near to the surface to communicate with the electrode) in the DS assay the RP can be bound to DNA regardless of its position and can used for the determination of the length of DNA repetitive sequences. Several fmols or about a hundred of amol of a RP with osmium-labeled (T)(50) tail can be detected at PGE and HMDE, respectively, at 1-2 min accumulation time.
在电化学DNA杂交分析中，常使用末端标记有电子活性化合物的靶标或探针DNA。我们表明，多个产生法拉第信号（在碳电极或汞电极上）和催化信号（在汞电极上）的锇标记可以很容易地共价连接到DNA分子上。我们使用（GAA）（7）（T）（n）寡脱氧核苷酸（ODN），其中n在5到50之间。（T）（n）尾巴用四氧化锇、2,2'-联吡啶进行选择性修饰，使（GAA）（7）重复序列保持完整以用于DNA杂交。这些ODN在DNA杂交双表面（DS）分析中用作报告探针（RP），使用磁珠进行DNA杂交，热解石墨（PGE）或悬汞滴（HMDE）电极进行电化学检测。我们表明，与通常的单表面方法（其中RP必须在靠近表面处与靶标DNA结合以与电极通信）不同，在DS分析中，RP可以与DNA结合而不管其位置如何，并且可用于测定DNA重复序列的长度。在1-2分钟的积累时间下，分别在PGE和HMDE上可以检测到几个飞摩尔或约一百个阿托摩尔带有锇标记（T）（50）尾巴的RP。
