Direct enzyme-amplified electrical recognition of a 30-base model oligonucleotide
Direct enzyme-amplified electrical recognition of a 30-base model oligonucleotide
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DOI：
10.1021/ja960490o
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发表时间：
1996-06-12
影响因子：
15
通讯作者：
Heller, A
中科院分区：
文献类型：
--
作者：
deLumleyWoodyear, T;Campbell, CN;Heller, A
电化学传感器基于插入钌和钴络合物 6、7 和有机染料 8、9 的电化学发光 1-5 和循环伏安法以及共价结合到单条 DNA 链 10 的氧化还原功能来识别 DNA 链的杂交。还描述了抗生物素蛋白-生物素缀合物的直接酶放大电流亲和力测定。 11 在该系统中，生物素化辣根过氧化物酶与共价附着于电子传导氧化还原水凝胶的抗生物素蛋白结合，导致酶的“布线”，即其与电极的电连接。这种连接产生与缀合物数量成正比的 H2O2 电还原电流。在这里，我们展示了模型寡核苷酸的杂交可以直接测量为电流。当互补链共价结合到水凝胶上时，H2O2 的连续电还原会产生电流，该水凝胶会通过寡核苷酸链的辣根过氧化物酶 (HRP) 标记进行电催化（图 1）。在这种水凝胶中，电子通过氧化还原聚合物网络的碰撞片段之间的电子自交换而扩散，这些片段在水合时变得可移动。图 12 将一条聚（脱氧胸苷）-5'-磷酸（pd (T) 25-30）寡核苷酸 I 链附着到玻璃碳电极上的聚丙烯酰胺基电子传导氧化还原水凝胶薄膜上。 13 互补链聚（脱氧腺苷）-5'-磷酸 (pd (A) 25-30)、寡核苷酸 II，用 HRP 标记。杂交后，酶被电连接至电极，并且 H2O2 被电催化还原。所得电流密度为 1.4（0.2 μA/cm2，在相对于 Ag/AgCl 处于 0.0 V 的电极上。HRP 与非互补寡核苷酸（例如 pd (T) 25-30 或聚（脱氧鸟苷）-5'-磷酸 (pd (G) 12-18)、寡核苷酸 III）非特异性结合所产生的电流密度仅为 0.07 (0.02 μA/cm2 是通过使用聚乙二醇二缩水甘油醚（Polysciences Cat# 08211）作为交联剂将共聚物 PAAPVI-Os（图 2a）与聚丙烯酰胺酰肼（Sigma，目录号 P-9905）（图 2b）交联而形成的电子传导氧化还原水凝胶。三种聚合物各自的重量分数为 61： 37: 2，寡核苷酸 I 通过碳二亚胺偶联与水凝胶的酰肼功能共价结合，如图 3 所示；1.85 x 10-4 g 的 pd (A) 25-30 用 3 x 10-3 L 的 0.1 M 处理。咪唑、0.15 M EDC、2.5×10-3 M 一水合肼溶液，23℃ 16 小时 15 为了除去过量的肼，将寡核苷酸在乙醇中沉淀，
Electrochemical sensors recognizing hybridization of DNA strands based on electrogenerated chemiluminescence1-5 and cyclic voltammetry of intercalated ruthenium and cobalt complexes6, 7 and organic dyes8, 9 and of redox functions covalently bound to single DNA strands10 have been reported. Also a direct enzyme-amplified amperometric affinity assay for the avidin-biotin conjugate has been described. 11 In this system, bonding of biotinylated horseradish peroxidase to avidin covalently attached to an electron-conducting redox hydrogel resulted in the “wiring” of the enzyme, ie its electrical connection to the electrode. Such connection produced an H2O2 electroreduction current proportional to the number of conjugates. Here we show that the hybridization of a model oligonucleotide can be directly measured as an electrical current. The current flows as a result of continuous electroreduction of H2O2, electrocatalyzed by the horseradish peroxidase (HRP) label of an oligonucleotide strand when the complementary strand is covalently bound to a hydrogel that electrically “wires” the HRP (Figure 1). In such a hydrogel, electrons diffuse via self-exchange of electrons between colliding segments of the redox polymer network, which become mobile when hydrated. 12 A strand of poly (deoxythymidine)-5′-phosphate (pd (T) 25-30), oligonucleotide I, was attached to a film of a polyacrylamidebased, electron-conducting redox hydrogel on a vitreous carbon electrode. 13 The complementary strand, poly (deoxyadenosine)-5′-phosphate (pd (A) 25-30), oligonucleotide II, was labeled with HRP. Upon hybridization the enzyme was electrically wired to the electrode, and H2O2 was electrocatalytically reduced. The resulting current density was 1.4 (0.2 μA/cm2 on an electrode poised at 0.0 V versus Ag/AgCl. The current density resulting from nonspecific binding of HRP bound to a noncomplementary oligonucleotide, such as pd (T) 25-30 or poly (deoxyguanosine)-5′-phosphate (pd (G) 12-18), oligonucleotide III, was only 0.07 (0.02 μA/cm2. The electron-conducting redox hydrogel was formed by crosslinking on a vitreous carbon electrode the copolymer PAAPVI-Os (Figure 2a) with polyacrylamide-hydrazide (Sigma, Cat# P-9905)(Figure 2b) using poly (ethylene glycol diglycidyl ether)(Polysciences Cat# 08211) as a cross-linker. The respective weight fractions of the three polymers were 61: 37: 2, and the total polymer loading was 0.4 mg cm-2. Oligonucleotide I was covalently bound by carbodiimide coupling to hydrazide functions of the hydrogel by a reported process. 14 Oligonucleotide II was HRP labeled, as shown in Figure 3; 1.85× 10-4 g of pd (A) 25-30 was treated with 3× 10-3 L of a 0.1 M imidazole, 0.15 M EDC, 2.5× 10-3 M hydrazine monohydrate solution at 23 C for 16 h. 15 To remove the excess hydrazine, the oligonucleotide was precipitated in ethanol,