Arockiam Siril, Nguyen Vi T, Knappenberger Mark, Anderson Clifford, Hansen Michael, Murugan Vel, Christen Jennifer Blain, Anderson Karen S
Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA, PO Box 876401.
Center for Bioelectronics and Biosensors, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA, PO Box 876401.
Anal Methods. 2025 May 29;17(21):4321-4333. doi: 10.1039/d4ay02201g.
Point-of-care technologies have become an essential tool in molecular diagnostics. Traditional laboratory-based nucleic acid extraction methods are laborious, costly, and hard to implement in point-of-care (POC) settings. POC nucleic acid extraction methods using silica membranes pose a significant technical challenge to simplify and efficiently extract RNA from biofluids such as saliva without degradation. Here, we have focused on addressing the POC nucleic acid (NA) extraction challenges by optimizing the RNase inactivation, viral lysis, NA binding conditions, and NA elution and adapting it to on-chip extraction. We have evaluated reducing agents, chaotropic salt (guanidine HCl), heat, proteinase K treatment, and elution buffer conditions. We have formulated POC-Pure, a cost-effective, efficient custom buffer-extraction method using silica to bind and release nucleic acids from salivary samples. To purify and concentrate NA, we have fabricated a microfluidic chip using xurographic and laser cutting techniques which incorporates a three-way valve actuator. We assessed the downstream application compatibility of the POC-Pure method using a POC-suitable loop-mediated isothermal amplification (LAMP). The simplified POC-Pure extraction method can purify and concentrate DNA and RNA with a lower limit of detection under 0.25 copies per μL and 0.5 copies per μL, respectively, using a 200 μL sample input. Thus, we have developed and demonstrated an on-chip nucleic acid extraction from saliva.
即时检测技术已成为分子诊断中的一项重要工具。传统的基于实验室的核酸提取方法费力、成本高，且难以在即时检测（POC）环境中实施。使用硅胶膜的POC核酸提取方法在简化并有效从唾液等生物流体中提取RNA且不使其降解方面面临重大技术挑战。在此，我们专注于通过优化核糖核酸酶失活、病毒裂解、核酸结合条件以及核酸洗脱，并使其适用于芯片上提取，来应对POC核酸（NA）提取挑战。我们评估了还原剂、离液盐（盐酸胍）、加热、蛋白酶K处理以及洗脱缓冲液条件。我们制定了POC - Pure方法，这是一种使用硅胶从唾液样本中结合并释放核酸的经济高效的定制缓冲液提取方法。为了纯化和浓缩NA，我们使用刻写和激光切割技术制造了一种包含三通阀致动器的微流控芯片。我们使用适用于POC的环介导等温扩增（LAMP）评估了POC - Pure方法的下游应用兼容性。简化的POC - Pure提取方法使用200μL样本输入，分别能以低于每微升0.25拷贝和每微升0.5拷贝的检测下限纯化和浓缩DNA和RNA。因此，我们开发并展示了一种从唾液中进行芯片上核酸提取的方法。
