Miodek Anna, Regan Edward M, Bhalla Nikhil, Hopkins Neal A E, Goodchild Sarah A, Estrela Pedro
Department of Electronic and Electrical Engineering, University of Bath, Bath BA2 7AY, UK.
Defence Science and Technology Laboratory, Porton Down, Salisbury, SP4 0JQ, UK.
Sensors (Basel). 2015 Sep 29;15(10):25015-32. doi: 10.3390/s151025015.
An aptasensor with enhanced anti-fouling properties has been developed. As a case study, the aptasensor was designed with specificity for human thrombin. The sensing platform was developed on screen printed electrodes and is composed of a self-assembled monolayer made from a ternary mixture of 15-base thiolated DNA aptamers specific for human thrombin co-immobilised with 1,6-hexanedithiol (HDT) and further passivated with 1-mercapto-6-hexanol (MCH). HDT binds to the surface by two of its thiol groups forming alkyl chain bridges and this architecture protects from non-specific attachment of molecules to the electrode surface. Using Electrochemical Impedance Spectroscopy (EIS), the aptasensor is able to detect human thrombin as variations in charge transfer resistance (Rct) upon protein binding. After exposure to a high concentration of non-specific Bovine Serum Albumin (BSA) solution, no changes in the Rct value were observed, highlighting the bio-fouling resistance of the surface generated. In this paper, we present the optimisation and characterisation of the aptasensor based on the ternary self-assembled monolayer (SAM) layer. We show that anti-fouling properties depend on the type of gold surface used for biosensor construction, which was also confirmed by contact angle measurements. We further studied the ratio between aptamers and HDT, which can determine the specificity and selectivity of the sensing layer. We also report the influence of buffer pH and temperature used for incubation of electrodes with proteins on detection and anti-fouling properties. Finally, the stability of the aptasensor was studied by storage of modified electrodes for up to 28 days in different buffers and atmospheric conditions. Aptasensors based on ternary SAM layers are highly promising for clinical applications for detection of a range of proteins in real biological samples.
一种具有增强抗污性能的适体传感器已被开发出来。作为一个案例研究，该适体传感器被设计为对人凝血酶具有特异性。传感平台是在丝网印刷电极上开发的，由一种自组装单分子层组成，该单分子层由对人凝血酶具有特异性的15碱基硫醇化DNA适体与1,6 - 己二硫醇（HDT）的三元混合物共同固定而成，并进一步用1 - 巯基 - 6 - 己醇（MCH）进行钝化处理。HDT通过其两个硫醇基团与表面结合形成烷基链桥，这种结构可防止分子非特异性附着到电极表面。使用电化学阻抗谱（EIS），该适体传感器能够检测人凝血酶，因为蛋白质结合时电荷转移电阻（Rct）会发生变化。在暴露于高浓度的非特异性牛血清白蛋白（BSA）溶液后，未观察到Rct值的变化，突出了所产生表面的抗生物污染性。在本文中，我们展示了基于三元自组装单分子层（SAM）层的适体传感器的优化和表征。我们表明，抗污性能取决于用于构建生物传感器的金表面类型，这也通过接触角测量得到了证实。我们进一步研究了适体与HDT之间的比例，该比例可以决定传感层的特异性和选择性。我们还报告了用于电极与蛋白质孵育的缓冲液pH值和温度对检测和抗污性能的影响。最后，通过在不同缓冲液和大气条件下将修饰电极储存长达28天，研究了适体传感器的稳定性。基于三元SAM层的适体传感器在临床应用中检测实际生物样品中的一系列蛋白质方面具有很大的前景。
