采用电活性标记物N-(2-乙基-二茂铁)马来酰亚胺检测与表面固定的 一致性双链DNA特异性作用的p53蛋白质

建立了电化学检测表面固定捕获的野生型p53蛋白质的方法. 首先在金电极表面形成巯基化的单链DNA探针/己硫醇(HT)混合自组装膜, 随后巯基化的单链DNA探针与溶液中序列匹配的靶点DNA杂交, 所形成的一致性双链DNA捕获溶液中的野生型p53蛋白质. p53分子表面的半胱氨酸残基采用巯基特异性试剂N-(2-乙基-二茂铁)马来酰亚胺(Fc-Mi)进行衍生. 通过检测二茂铁的电化学信号来指示p53与一致性双链DNA之间的特异性相互作用. p53蛋白质与双链DNA的键合程度取决于双链DNA的序列. 该方法可检测的p53最低浓度为1.33 nmol•L－1.

Voltammetric Detection of p53 at Electrodes Modified with Consensus Double-Stranded Oligonucleotides with N-(2-Ferrocenylethyl)maleimide

A voltammetric method for the determination of surface-confined wild-type p53 protein has been developed. Consensus double-stranded oligonucleotide (ds-DNA) was first formed onto gold electrodes via hybridization of thiolated single-stranded DNA (ss-DNA)/hexanethiol (HT) mixed self-assembled monolayer with the complementary target DNA. The resultant consensus ds-DNA was subsequently used to capture wild-type p53 in solution. The cysteine residues on the exterior of the p53 molecules were derivatized with a thiol-specific reagent, N-(2-ferrocenylethyl)maleimide (Fc-Mi). Well-defined voltammetric peaks that report on the interaction of p53 with the consensus ds-DNA were obtained. The level of p53 binding was found to be dependent on the sequence of the double-stranded (ds-DNA). The present method can measure p53 concentration as low as 1.33 nmol•L－1.