Department of Biomedical Engineering, The University of Arizona, Tucson, AZ, 85721, United States.
Wyant College of Optical Sciences, The University of Arizona, Tucson, AZ, 85721, United States.
Biosens Bioelectron. 2025 Jan 1;267:116861. doi: 10.1016/j.bios.2024.116861. Epub 2024 Oct 25.
This work demonstrates a novel, non-fluorescence approach to the length identification of polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA) products, utilizing capillary flow velocities on paper microfluidic chips. It required only a blank paper chip, aminated microspheres, and a smartphone, with a rapid assay time and under ambient lighting. A smartphone evaluated the initial capillary flow velocities on the paper chips for the PCR and RPA products from various bacterial samples, where the pre-loaded aminated microspheres differentiated their flow velocities. Flow velocities were analyzed at different time frames and compared with the instantaneous flow velocities and interfacial tension (γ) data. Subsequent error analysis justified the use of the initial time frames. A robust linear relationship could be established between the initial flow velocities against the square root of the product lengths, with R values of 0.981 for PCR and 0.993 for RPA. The assay seemed not to have a significant dependency on the cycle numbers and initial target concentrations. This novel method can be potentially used with various paper microfluidic methods of nucleic acid amplification tests towards rapid and handheld assays.
这项工作展示了一种新颖的非荧光方法，用于通过纸微流控芯片上的毛细流速来识别聚合酶链反应（PCR）和重组酶聚合扩增（RPA）产物的长度。它只需要空白的纸芯片、氨基化微球和智能手机，具有快速检测时间和环境光照条件。智能手机评估了来自各种细菌样本的 PCR 和 RPA 产物在纸芯片上的初始毛细流速，其中预加载的氨基化微球可区分它们的流速。在不同的时间框架下分析流速，并与瞬时流速和界面张力（γ）数据进行比较。随后的误差分析证明了使用初始时间框架的合理性。可以在初始流速与产物长度的平方根之间建立稳健的线性关系，PCR 的 R 值为 0.981，RPA 的 R 值为 0.993。该测定似乎不受循环次数和初始靶浓度的显著影响。这种新方法可与各种用于核酸扩增测试的纸微流控方法结合使用，以实现快速和手持式检测。
