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Electrochemical genosensor for Klotho detection based on aliphatic and aromatic thiols self-assembled monolayers.
Talanta ( IF 5.6 ) Pub Date : 2020-01-11 , DOI: 10.1016/j.talanta.2020.120735 M Sánchez-Paniagua 1 , S Palenzuela-Batista 1 , C L Manzanares-Palenzuela 2 , B López-Ruiz 1
Talanta ( IF 5.6 ) Pub Date : 2020-01-11 , DOI: 10.1016/j.talanta.2020.120735 M Sánchez-Paniagua 1 , S Palenzuela-Batista 1 , C L Manzanares-Palenzuela 2 , B López-Ruiz 1
Affiliation
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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 5·10-10 to 5·10-8 M was obtained with CP/HDT/MCH monolayer, with a detection limit of 0.5 nM and RSD of 8.10%.
中文翻译：
用于脂族和芳族硫醇自组装单分子层的克洛索检测的电化学基因传感器。
Klotho基因表达的变化与慢性肾脏疾病有关，其作为早期生物标志物的潜力正在研究中。我们首次报告通过生物传感器平台检测Klotho基因。自组装混合单层（SAMs）作为丝网印刷金电极中的DNA固定方法和夹心格式检测被用于开发电化学基因传感器以检测100聚体DNA片段， mRNA Klotho基因。通过循环伏安法和电化学阻抗光谱法评估了基于脂肪族（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作为感测相。研究了多种配置，更改组分添加的顺序，并比较共固定和两步固定过程。发现二元SAM制备过程由依次添加硫醇稀释剂和CP组成，对电化学性能的影响最小。与脂族SAM的值相比，硫代芳族二元DNA单层MPPA / CP的信噪比显着提高。由MCH和HDT形成的三元单分子层具有良好的分数覆盖水平，并在表面产生更多可逆的氧化还原反应，主要是在CP首次被固定时，即CP / HDT / MCH。与二元SAM相比，使用三元SAM可以显着减少空白信号和非特异性（非互补序列）信号，并增加2倍。与MPAA / CP SAM相比，S / B比（目标的10 nM）的42倍。使用CP / HDT / MCH单层，可得到5·10-10至5·10-8 M的线性响应，检测限为0.5 nM，RSD为8.10％。
更新日期：2020-01-13
中文翻译：
用于脂族和芳族硫醇自组装单分子层的克洛索检测的电化学基因传感器。
Klotho基因表达的变化与慢性肾脏疾病有关，其作为早期生物标志物的潜力正在研究中。我们首次报告通过生物传感器平台检测Klotho基因。自组装混合单层（SAMs）作为丝网印刷金电极中的DNA固定方法和夹心格式检测被用于开发电化学基因传感器以检测100聚体DNA片段， mRNA Klotho基因。通过循环伏安法和电化学阻抗光谱法评估了基于脂肪族（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作为感测相。研究了多种配置，更改组分添加的顺序，并比较共固定和两步固定过程。发现二元SAM制备过程由依次添加硫醇稀释剂和CP组成，对电化学性能的影响最小。与脂族SAM的值相比，硫代芳族二元DNA单层MPPA / CP的信噪比显着提高。由MCH和HDT形成的三元单分子层具有良好的分数覆盖水平，并在表面产生更多可逆的氧化还原反应，主要是在CP首次被固定时，即CP / HDT / MCH。与二元SAM相比，使用三元SAM可以显着减少空白信号和非特异性（非互补序列）信号，并增加2倍。与MPAA / CP SAM相比，S / B比（目标的10 nM）的42倍。使用CP / HDT / MCH单层，可得到5·10-10至5·10-8 M的线性响应，检测限为0.5 nM，RSD为8.10％。
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