Zimmers Zackary A, Boyd Alexander D, Stepp Hannah E, Adams Nicholas M, Haselton Frederick R
Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37212, USA.
Department of Chemistry, Vanderbilt University, Nashville, TN 37212, USA.
Micromachines (Basel). 2021 Sep 30;12(10):1204. doi: 10.3390/mi12101204.
Among nucleic acid diagnostic strategies, non-enzymatic tests are the most promising for application at the point of care in low-resource settings. They remain relatively under-utilized, however, due to inadequate sensitivity. Inspired by a recent demonstration of a highly-sensitive dumbbell DNA amplification strategy, we developed an automated, self-contained assay for detection of target DNA. In this new diagnostic platform, called the automated Pi-powered looping oligonucleotide transporter, magnetic beads capture the target DNA and are then loaded into a microfluidic reaction cassette along with the other reaction solutions. A stepper motor controls the motion of the cassette relative to an external magnetic field, which moves the magnetic beads through the reaction solutions automatically. Real-time fluorescence is used to measure the accumulation of dumbbells on the magnetic bead surface. Left-handed DNA dumbbells produce a distinct signal which reflects the level of non-specific amplification, acting as an internal control. The autoPiLOT assay detected as little as 5 fM target DNA, and was also successfully applied to the detection of DNA. The autoPiLOT design is a novel step forward in the development of a sensitive, user-friendly, low-resource, non-enzymatic diagnostic test.
在核酸诊断策略中，非酶检测在资源匮乏地区的即时检测应用方面最具前景。然而，由于灵敏度不足，它们的利用率仍然相对较低。受最近一项高灵敏度哑铃状DNA扩增策略演示的启发，我们开发了一种用于检测目标DNA的自动化、独立式检测方法。在这个名为自动Pi驱动的环状寡核苷酸转运体的新诊断平台中，磁珠捕获目标DNA，然后与其他反应溶液一起加载到微流控反应盒中。步进电机控制反应盒相对于外部磁场的运动，外部磁场使磁珠自动穿过反应溶液。实时荧光用于测量磁珠表面哑铃状分子的积累。左手性DNA哑铃状分子产生独特的信号，反映非特异性扩增水平，作为内部对照。自动PiLOT检测方法可检测低至5 fM的目标DNA，并且也成功应用于DNA的检测。自动PiLOT设计是开发灵敏、用户友好、资源匮乏型非酶诊断测试的一个新的进步。
