The preparation of an integrated biosensor for the easy, fast, and sensitive determination of miRNAs is described based on a direct hybridization format and a label-free voltammetric detection. The biosensor involves a disposable carbon electrode substrate doubly nanostructured with reduced graphene oxide (rGO) and AuNPs modified with pyrene carboxylic acid (PCA) and 6-ferrocenylhexanethiol (Fc-SH), respectively. A synthetic amino terminated DNA capture probe was covalently immobilized on the CO 2 H moieties of PCA/rGO, while Fc-SH was used as a signaling molecule. Differential pulse voltammetry was employed to record the decrease in the oxidation peak current of Fc after the hybridization due to the hindering of the electron transfer upon the formation of the DNA-RNA duplex on the electrode surface. The stepwise biosensor preparation was characterized by surface and electrochemical techniques showing the role played by each biosensor component as well as the reliability of the target miRNA determination. The determination of the oncogene miRNA-21 synthetic target allowed quantification in the low femtomolar range (LOD of 5 fM) with a high discrimination of single-base mismatched sequences in a single 30-min incubation step. The bioplatform allowed the determination of the target miRNA in a small amount of total RNA extracted from breast cancer (BC) cells or directly in serum samples collected from BC patients without the need for prior extraction, purification, amplification, or reverse transcription of the genetic material and with no matrix effect. Graphical abstract

中文翻译：

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使用无标记和无酶伏安生物传感器在 30 分钟的单个步骤中测定癌症患者血清中的 miRNA

基于直接杂交格式和无标记伏安检测描述了用于简单、快速和灵敏地测定 miRNA 的集成生物传感器的制备。该生物传感器包括一个一次性碳电极基底，其双重纳米结构由还原氧化石墨烯 (rGO) 和分别用芘羧酸 (PCA) 和 6-二茂铁基己硫醇 (Fc-SH) 修饰的 AuNPs 构成。合成的氨基末端 DNA 捕获探针共价固定在 PCA/rGO 的 CO 2 H 部分，而 Fc-SH 用作信号分子。采用微分脉冲伏安法记录杂交后由于在电极表面形成 DNA-RNA 双链体时电子转移受阻导致 Fc 氧化峰电流的降低。逐步生物传感器制备的特点是表面和电化学技术显示每个生物传感器组件所起的作用以及目标 miRNA 测定的可靠性。致癌基因 miRNA-21 合成靶标的确定允许在低飞摩尔范围（5 fM 的 LOD）中进行量化，并在单个 30 分钟的孵育步骤中高度区分单碱基错配序列。该生物平台允许在从乳腺癌 (BC) 细胞中提取的少量总 RNA 或直接在从 BC 患者收集的血清样本中测定目标 miRNA，而无需事先提取、纯化、扩增或逆转录遗传基因。材料，无基质效应。图形概要 致癌基因 miRNA-21 合成靶标的确定允许在低飞摩尔范围（5 fM 的 LOD）中进行量化，并在单个 30 分钟的孵育步骤中高度区分单碱基错配序列。该生物平台允许在从乳腺癌 (BC) 细胞中提取的少量总 RNA 或直接在从 BC 患者收集的血清样本中测定目标 miRNA，而无需事先提取、纯化、扩增或逆转录遗传基因。材料，无基质效应。图形概要 致癌基因 miRNA-21 合成靶标的确定允许在低飞摩尔范围（5 fM 的 LOD）中进行量化，并在一个 30 分钟的孵育步骤中高度区分单碱基错配序列。该生物平台允许在从乳腺癌 (BC) 细胞中提取的少量总 RNA 或直接在从 BC 患者收集的血清样本中测定目标 miRNA，而无需事先提取、纯化、扩增或逆转录遗传基因。材料，无基质效应。图形概要 该生物平台允许在从乳腺癌 (BC) 细胞提取的少量总 RNA 中或直接在从 BC 患者收集的血清样本中测定目标 miRNA，而无需事先提取、纯化、扩增或逆转录遗传基因。材料，无基质效应。图形概要 该生物平台允许在从乳腺癌 (BC) 细胞中提取的少量总 RNA 或直接在从 BC 患者收集的血清样本中测定目标 miRNA，而无需事先提取、纯化、扩增或逆转录遗传基因。材料，无基质效应。图形概要