Micro/Bio/Nanofluidics Unit, Okinawa Institute of Science and Technology, Onna, Okinawa, 904-0495, Japan; Institute of Mechanical Process Engineering and Mechanics, Applied Mechanics Group, Karlsruhe Institute of Technology, 76137, Karlsruhe, Germany.
Micro/Bio/Nanofluidics Unit, Okinawa Institute of Science and Technology, Onna, Okinawa, 904-0495, Japan.
Biosens Bioelectron. 2019 Oct 1;142:111528. doi: 10.1016/j.bios.2019.111528. Epub 2019 Jul 23.
DNA polymerase catalyzes the replication of DNA, one of the key steps in cell division. The control and understanding of this reaction owns great potential for the fundamental study of DNA-enzyme interactions. In this context, we developed a label-free microfluidic biosensor platform based on the principle of localized surface plasmon resonance (LSPR) to detect the DNA-polymerase reaction in real-time. Our microfluidic LSPR chip integrates a polydimethylsiloxane (PDMS) channel bonded with a nanoplasmonic substrate, which consists of densely packed mushroom-like nanostructures with silicon dioxide stems (40 nm) and gold caps (22 nm), with an average spacing of 19 nm. The LSPR chip was functionalized with single-stranded DNA (ssDNA) template (T30), spaced with hexanedithiol (HDT) in a molar ratio of 1:1. The DNA primer (P8) was then attached to T30, and the second strand was subsequently elongated by DNA polymerase assembling nucleotides from the surrounding fluid. All reaction steps were detected in-situ inside the microfluidic LSPR chip, at room temperature, in real-time, and label-free. In addition, the sensor response was successfully correlated with the amount of DNA and HDT molecules immobilized on the LSPR sensor surface. Our platform represents a benchmark in developing microfluidic LSPR chips for DNA-enzyme interactions, further driving innovations in biosensing technologies.
DNA 聚合酶催化 DNA 的复制，这是细胞分裂的关键步骤之一。控制和理解这种反应对于研究 DNA-酶相互作用具有很大的潜力。在这种情况下，我们开发了一种基于局域表面等离子体共振（LSPR）原理的无标记微流控生物传感器平台，用于实时检测 DNA-聚合酶反应。我们的微流控 LSPR 芯片集成了一个聚二甲基硅氧烷（PDMS）通道，该通道与纳米等离子体基底结合，纳米等离子体基底由具有二氧化硅茎（约 40nm）和金帽（约 22nm）的密集蘑菇状纳米结构组成，平均间距为 19nm。LSPR 芯片用单链 DNA（ssDNA）模板（T30）功能化，T30 与 1:1 摩尔比的己二硫醇（HDT）间隔开。然后将 DNA 引物（P8）连接到 T30 上，随后由 DNA 聚合酶从周围流体中组装核苷酸来延伸第二条链。所有反应步骤都在微流控 LSPR 芯片内部，在室温下，实时，无标记地进行原位检测。此外，传感器响应与固定在 LSPR 传感器表面上的 DNA 和 HDT 分子的数量成功相关。我们的平台代表了开发用于 DNA-酶相互作用的微流控 LSPR 芯片的基准，进一步推动了生物传感技术的创新。
