Cavallo Francesca Romana, Mirza Khalid B, de Mateo Sara, Nikolic Konstantin, Rodriguez-Manzano Jesus, Toumazou Christofer
Centre for Bio-Inspired Technology, Imperial College London, London SW7 2AZ, United Kingdom.
Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela, Odisha 769008, India.
ACS Sens. 2021 Mar 26;6(3):709-715. doi: 10.1021/acssensors.0c02605. Epub 2021 Mar 2.
Protein quantification is traditionally performed through enzyme-linked immunosorbent assay (ELISA), which involves long preparation times. To overcome this, new approaches use aptamers as an alternative to antibodies. In this paper, we present a new approach to quantify proteins with short DNA aptamers through polymerase chain reaction (PCR) resulting in shorter protocol times with comparatively improved limits of detection. The proposed method includes a novel way to quantify both the target protein and the corresponding short DNA-aptamers simultaneously, which also allows us to fully characterize the performance of aptasensors. Human leptin is used as a target protein to validate this technique, because it is considered an important biomarker for obesity-related studies. In our experiments, we achieved the lowest limit of detection of 100 pg/mL within less than 2 h, a limit affected by the dissociation constant of the leptin aptamer, which could be improved by selecting a more specific aptamer. Because of the simple and inexpensive approach, this technique can be employed for Lab-On-Chip implementations and for rapid "on-site" quantification of proteins.
蛋白质定量传统上是通过酶联免疫吸附测定（ELISA）进行的，这需要很长的准备时间。为了克服这一问题，新方法使用适体作为抗体的替代品。在本文中，我们提出了一种通过聚合酶链反应（PCR）用短DNA适体定量蛋白质的新方法，从而缩短了实验方案时间，并相对提高了检测限。所提出的方法包括一种同时定量目标蛋白质和相应短DNA适体的新方法，这也使我们能够全面表征适体传感器的性能。人瘦素用作目标蛋白质来验证该技术，因为它被认为是肥胖相关研究的重要生物标志物。在我们的实验中，我们在不到2小时内实现了最低检测限为100 pg/mL，该检测限受瘦素适体解离常数的影响，通过选择更特异的适体可以得到改善。由于该方法简单且成本低廉，因此可用于芯片实验室应用以及蛋白质的快速“现场”定量。
