Rapid on-chip nucleic acid extraction for point-of-care salivary diagnostics

Abstract

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.