Interfibio Research Group, Department of Chemical Engineering, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain.
Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys 23, 08010, Barcelona, Spain.
Mikrochim Acta. 2021 Mar 9;188(4):117. doi: 10.1007/s00604-021-04771-8.
A reagent-less DNA sensor has been developed exploiting a combination of gold nanoparticles, modified primers, and isothermal amplification. It is applied to the determination ofKarlodinium armiger, a toxic microalgae, as a model analyte to demonstrate this generic platform. Colloidal gold nanoparticles with an average diameter of 14 ± 0.87 nm were modified with a mixed self-assembled monolayer of thiolated 33-mer DNA probes and (6-mercaptohexyl) ferrocene. Modified primers, exploiting a C3 spacer between the primer-binding site and an engineered single-stranded tail, were used in an isothermal recombinase polymerase amplification reaction to produce an amplicon by two single-stranded tails. These tails were designed to be complementary to a gold electrode tethered capture oligo probe, and an oligo probe immobilized on the gold nanoparticles, respectively. The time required for hybridization of the target tailed DNA with the surface immobilized probe and reporter probe immobilized on AuNPs was optimized and reduced to 10 min, in both cases. Amplification time was further optimized to be 40 min to ensure the maximum signal. Under optimal conditions, the limit of detection was found to be 1.6 fM of target dsDNA. Finally, the developed biosensor was successfully applied to the detection of genomic DNA extracted from a seawater sample that had been spiked with K. armiger cells. The demonstrated generic electrochemical genosensor can be exploited for the detection of any DNA sequence and ongoing work is moving towards an integrated system for use at the point-of-need.
已经开发出一种无试剂 DNA 传感器，该传感器利用金纳米粒子、修饰引物和等温扩增的组合。将其应用于鉴定毒性微藻卡鲁藻作为模型分析物，以证明这种通用平台。平均直径为 14±0.87nm 的胶体金纳米粒子用巯基修饰的 33 聚体 DNA 探针和（6-巯基己基）二茂铁的混合自组装单层进行修饰。修饰引物利用引物结合位点和工程化的单链尾巴之间的 C3 间隔物，用于等温重组酶聚合酶扩增反应，通过两条单链尾巴产生扩增子。这些尾巴设计为与固定在金电极上的捕获寡核苷酸探针以及固定在金纳米粒子上的寡核苷酸探针互补。目标加尾 DNA 与表面固定探针和固定在 AuNPs 上的报告探针杂交的时间被优化并缩短至 10 分钟，在这两种情况下都是如此。进一步优化扩增时间以达到 40 分钟，以确保最大信号。在最佳条件下，发现目标 dsDNA 的检测限为 1.6 fM。最后，开发的生物传感器成功应用于检测从已添加卡鲁藻细胞的海水样品中提取的基因组 DNA。所展示的通用电化学生物传感器可用于检测任何 DNA 序列，正在进行的工作正在朝着用于即时需求的集成系统发展。
