Gore Mitchell R, Szalai Veronika A, Ropp Patricia A, Yang Ivana V, Silverman Joel S, Thorp H Holden
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, USA.
Anal Chem. 2003 Dec 1;75(23):6586-92. doi: 10.1021/ac034918v.
The electrochemical detection of nucleic acid targets at low concentrations has a number of applications in diagnostics and pharmaceutical research. Self-assembled monolayers of alkanethiol-derivatized oligonucleotides on gold electrodes provide a useful platform for such detectors, and the electrocatalytic oxidation of nucleobases included in the DNA targets is a particularly sensitive method of electrochemical detection. A strategy has been developed for combining these two aspects by substituting either 7,8-dihydro-8-oxoguanine (8G) or 5-aminouridine (5U) into DNA targets. Upon hybridization of targets containing these modified nucleobases, electrocatalytic signals at probe-modified gold electrodes are observed in the presence of Os(bpy)(3)(2+), which oxidizes both 8G and 5U upon oxidation to the Os(III) state. Self-assembled monolayers were prepared on both macro (1.6 mm) and micro (25 microm) gold electrodes using published procedures involving C6-terminated alkanethiol oligonucleotides and mercaptohexanol as the diluent. The extent of electrode modification by the modified probe was assessed using radiolabeling and a standard chronocoulometry method; both approaches gave loading levels within expected ranges ((1-6) x 10(12) molecules/cm(2)). Hybridization of the modified targets where the non-native nucleobase was incorporated by solid-phase synthesis produced electrocatalytic signals from strands that were independently detected using radiolabeling and chronocoulometry. This result was used as a basis to develop an on-electrode amplification scheme where Taq polymerase was used to extend the immobilized DNA probes from solution-phase polymeric templates using modified nucleotriphosphates. This reaction produced an electrode that was modified with extended DNA containing the appropriate modified nucleotide. Radiolabeled nucleotide triphosphates were used to confirm the desired on-electrode DNA synthesis. When these electrodes were cycled in the presence of Os(bpy)(3)(2+), electrocatalytic signals were observed when as little as 40 amol (400 fM) of the desired target was present in the hybridization solution.
低浓度核酸靶标的电化学检测在诊断和药物研究中有许多应用。金电极上烷硫醇衍生化寡核苷酸的自组装单层为这类检测器提供了一个有用的平台，DNA靶标中所含核碱基的电催化氧化是一种特别灵敏的电化学检测方法。已经开发出一种策略，通过将7,8 - 二氢 - 8 - 氧代鸟嘌呤（8G）或5 - 氨基尿苷（5U）取代到DNA靶标中来结合这两个方面。当含有这些修饰核碱基的靶标杂交时，在存在Os(bpy)(3)(2+)的情况下，在探针修饰的金电极上观察到电催化信号，Os(bpy)(3)(2+)在氧化成Os(III)态时会氧化8G和5U。使用已发表的程序，以C6端烷硫醇寡核苷酸和巯基己醇作为稀释剂，在宏观（1.6毫米）和微观（25微米）金电极上制备自组装单层。使用放射性标记和标准计时库仑法评估修饰探针的电极修饰程度；两种方法得到的负载水平都在预期范围内（(1 - 6)×10(12)个分子/平方厘米）。通过固相合成掺入非天然核碱基的修饰靶标的杂交产生了来自链的电催化信号，这些链使用放射性标记和计时库仑法独立检测。该结果被用作开发一种电极上扩增方案的基础，其中使用Taq聚合酶从溶液相聚合物模板使用修饰的三磷酸核苷酸延伸固定的DNA探针。该反应产生了一个用含有适当修饰核苷酸的延伸DNA修饰的电极。放射性标记的三磷酸核苷酸用于确认所需的电极上DNA合成。当这些电极在存在Os(bpy)(3)(2+)的情况下循环时，当杂交溶液中存在低至40 amol（400 fM）的所需靶标时，观察到电催化信号。
