One-step conjugation of aminoferrocene to phosphate groups as electroactive probes for electrochemical detection of sequence-specific DNA.
One-step conjugation of aminoferrocene to phosphate groups as electroactive probes for electrochemical detection of sequence-specific DNA.
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DOI：
10.1016/j.bios.2014.10.015
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发表时间：
2015-03
影响因子：
12.6
通讯作者：
Qiong Hu;Xianbao Deng;Xuehua Yu;J. Kong;Xueji Zhang
中科院分区：
文献类型：
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作者：
Qiong Hu;Xianbao Deng;Xuehua Yu;J. Kong;Xueji Zhang
首次证明了一种基于利用有机金属化合物氨基二茂铁 (AFC) 作为电活性探针的直接电化学 DNA 生物传感方法，其中探针只需在 1-乙基-3-(3-二甲基氨基丙基)碳二亚胺 (EDC) 和咪唑存在下通过一步缀合即可直接标记到杂交 PNA/DNA 异源双链体的游离磷酸基团。简而言之，首先将带有巯基的肽核酸（PNA）固定在金电极上，并用作捕获探针来特异性识别目标单链DNA（ssDNA）。杂交后，AFC探针直接标记到杂交的PNA/DNA异源双链体的游离5'端磷酸基团上，该磷酸基团被EDC和咪唑激活，然后将它们用作电活性探针来监测杂交。由于捕获的单链DNA以1:1的化学计量比用AFC标记，因此基于微分脉冲伏安法（DPV）对按比例标记的AFC进行电化学分析可以定量测定序列特异性DNA。在最佳条件下，该方法在0.1 nM至100 nM范围内，电流强度与ssDNA浓度对数之间呈现良好的线性关系，检测限为93 pM，在血清样品中具有令人满意的分析性能。此外，它对单核苷酸多态性（SNP）表现出优异的特异性，并排除了复杂的实验方案。更重要的是，这种方法的简单性及其与标准微制造技术的兼容性使其在实际应用中具有巨大潜力，特别是在首选简单程序的微阵列领域。
A straightforward electrochemical DNA biosensing approach based on exploiting organometallic compound, aminoferrocene (AFC), as electroactive probes was firstly demonstrated, where the probes could be directly labeled to the free phosphate groups of the hybridized PNA/DNA heteroduplexes merely through one-step conjugation in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and imidazole. Briefly, mercapto-terminated peptide nucleic acid (PNA) was firstly immobilized onto gold electrode and used as the capture probes for the specific recognition of target single-stranded DNA (ssDNA). After hybridization, AFC probes were directly labeled to the free 5′-terminal phosphate groups, which were activated by EDC and imidazole, of the hybridized PNA/DNA heteroduplexes, and then they were exploited as the electroactive probes to monitor the hybridization. As the captured ssDNA was labeled with AFC in the stoichiometric ratio of 1:1, thus the electrochemical analysis of the proportionally labeled AFC based on differential pulse voltammetry (DPV) enabled a quantitative determination of sequence-specific DNA. Under optimal conditions, the approach presented a good linear relationship between the current intensities and logarithm of ssDNA concentrations in the range from 0.1 nM to 100 nM with a detection limit of 93 pM, and it rendered satisfactory analytical performance in serum samples. Furthermore, it exhibited excellent specificity toward single-nucleotide polymorphism (SNP) and precluded complicated protocols. More importantly, the simplicity of this approach together with its compatibility with standard micro-fabrication techniques makes it great potential in practical applications, especially in microarray areas where simple procedures are preferred.