Departamento Química en Ciencias Farmacéuticas, Facultad de Farmacia, Universidad Complutense de Madrid, 28040, Madrid, Spain.
Center for Advanced Functional Nanorobots, Department of Inorganic Chemistry, University of Chemistry and Technology Prague, Technická 5, 166 28, Czech Republic.
Talanta. 2020 May 15;212:120735. doi: 10.1016/j.talanta.2020.120735. Epub 2020 Jan 11.
Changes in expression of Klotho gene are associated with chronic kidney disease and its potential as early biomarker is being studied. We report, for the first time, the detection of Klotho gene by a biosensor platform. Self-assembled mixed monolayers (SAMs) as DNA immobilization method in screen-printed gold electrodes and a sandwich format detection were used in the development of an electrochemical genosensor for the detection of a 100-mer DNA fragment, copy of the partial region of the mRNA Klotho gene. The use of different binary and ternary SAMs based on aliphatic (mercaptohexanol, MCH, and hexanedithiol, HDT) and aromatic (mercaptophenylacetic acid, MPAA) thiol diluents and capture probe (CP) as sensing phases was evaluated by cyclic voltammetry and electrochemical impedance spectroscopy. Multiple configurations were studied, changing the order of component addition and comparing co-immobilization and two-step immobilization processes. The procedure for binary SAM preparation consisting of sequential addition of a thiol diluent followed by CP was found to have the least detrimental impact on electrochemical performance. The signal-to-blank ratios increased considerably in the case of thioaromatic binary DNA monolayers, MPPA/CP, compared to the values obtained for aliphatic SAMs. Ternary monolayers formed by MCH and HDT rendered good fractional coverage levels and generated more reversible redox reactions at the surface, mostly when CP was firstly immobilized, CP/HDT/MCH. A significant reduction of the blank and non-specific (non-complementary sequence) signals was obtained with this ternary SAM, compared to binary SAMs and an increase of 2.42-fold of the S/B ratio (10 nM of target) compared with MPAA/CP SAMs. A linear response in the range of 5·10 to 5·10 M was obtained with CP/HDT/MCH monolayer, with a detection limit of 0.5 nM and RSD of 8.10%.
Klotho 基因表达的变化与慢性肾脏病有关，其作为早期生物标志物的潜力正在研究中。我们首次报告了生物传感器平台检测 Klotho 基因。自组装混合单层（SAMs）作为 DNA 固定方法在丝网印刷金电极上，并采用三明治结构检测，用于开发电化学基因传感器，以检测 100 -mer DNA 片段，即 Klotho 基因 mRNA 部分区域的副本。通过循环伏安法和电化学阻抗谱评估了基于脂肪族（巯基己醇（MCH）和己二硫醇（HDT）和芳香族（巯基苯乙酸（MPAA）硫醇稀释剂和捕获探针（CP）的不同二元和三元 SAM 的使用情况。改变组件添加顺序并比较共固定和两步固定过程，研究了多种配置。发现由依次添加硫醇稀释剂然后添加 CP 组成的二元 SAM 制备程序对电化学性能的不利影响最小。与脂肪族 SAM 相比，硫代芳香族二元 DNA 单层 MPPA/CP 的信号与空白比显着增加。由 MCH 和 HDT 形成的三元单层具有良好的分数覆盖率水平，并在表面产生更可逆的氧化还原反应，主要是当 CP 首先固定时，CP/HDT/MCH。与二元 SAM 相比，这种三元 SAM 可显着降低空白和非特异性（非互补序列）信号，并将 S/B 比值（10 nM 靶标）提高 2.42 倍，与 MPAA/CP SAM 相比。CP/HDT/MCH 单层在 5·10 至 5·10 M 的范围内获得线性响应，检测限为 0.5 nM，RSD 为 8.10%。
