Currently, electrochemical nucleic acid-based biosensing methodologies involving hybridization assays, specific recognition of RNA/DNA and RNA/RNA duplexes, and amplification systems provide an attractive alternative to conventional quantification strategies for the routine determination of relevant nucleic acids at different settings. A particularly relevant objective in the development of such nucleic acid biosensors is the design of as many as possible affordable, quick, and simple methods while keeping the required sensitivity. With this aim in mind, this work reports, for the first time, a thorough comparison between 11 methodologies that involve different assay formats and labeling strategies for targeting the same DNA. The assayed approaches use conventional sandwich and competitive hybridization assays, direct hybridization coupled to bioreceptors with affinity for RNA/DNA duplexes, multienzyme labeling bioreagents, and DNA concatamers. All of them have been implemented on the surface of magnetic beads (MBs) and involve amperometric transduction at screen-printed carbon electrodes (SPCEs). The influence of the formed duplex length and of the labeling strategy have also been evaluated. Results demonstrate that these strategies can provide very sensitive methods without the need for using nanomaterials or polymerase chain reaction (PCR). In addition, the sensitivity can be tailored within several orders of magnitude simply by varying the bioassay format, hybrid length or labeling strategy. This comparative study allowed us to conclude that the use of strategies involving longer hybrids, the use of antibodies with specificity for RNA/DNA heteroduplexes and labeling with bacterial antibody binding proteins conjugated with multiple enzyme molecules, provides the best sensitivity.

中文翻译：

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既不使用纳米材料也不使用核酸扩增的高灵敏度电化学核酸生物传感器开发不同策略的比较

当前，涉及杂交测定，RNA / DNA和RNA / RNA双链体的特异性识别以及扩增系统的基于电化学核酸的生物传感方法为常规测定不同设置的相关核酸提供了一种替代常规定量策略的有吸引力的选择。在这种核酸生物传感器的开发中，特别相关的目标是设计尽可能多的负担得起的，快速的和简单的方法，同时保持所需的灵敏度。出于这个目的，这项工作首次报告了11种方法的全面比较，这些方法涉及不同的测定形式和针对同一DNA的标记策略。被分析的方法使用常规的夹心和竞争性杂交分析，直接杂交与对RNA / DNA双链体，多酶标记生物试剂和DNA辅酶具有亲和力的生物受体偶联。所有这些都已在磁珠（MBs）的表面上实现，并且涉及丝网印刷碳电极（SPCE）上的安培转导。还评估了形成的双链体长度和标记策略的影响。结果表明，这些策略可以提供非常敏感的方法，而无需使用纳米材料或聚合酶链反应（PCR）。此外，只需更改生物测定形式，杂交长度或标记策略，即可在几个数量级内调整灵敏度。这项比较研究使我们可以得出结论，即使用涉及更长杂种的策略，