Gravity-driven millifluidic platform for magnetic solid-phase extraction of Enterocytozoon hepatopenaei DNA from
Xianlong Shi1, Yirong Chen1, Quan Sun2
1Key Laboratory of Luminescence Analysis and Molecular Sensing, Ministry of Education, Institute for Clean Energy and Advanced Materials, School of Materials and Energy, Southwest University, Chongqing, 400715, China.
Abstract:
Effective detection of Enterocytozoon hepatopenaei (EHP) in aquaculture is currently hindered by the lack of field-deployable extraction methods capable of processing complex, inhibitor-rich hepatopancreatic tissue. This study presents a gravity-driven millifluidic platform for the rapid extraction of EHP genomic DNA using an optimized, surfactant-compatible magnetic solid-phase extraction (MSPE) chemistry. Utilizing 5% PEG 8000 and 2.0 M NaCl, the platform facilitates the selectively capture of DNA from inhibitor-rich crustacean lysates. The 3D-printed device employs a tilting rocking plate to generate passive, gravity-driven flow, maintaining homogeneous magnetic bead suspension and maximizing solid-phase capture efficiency without external pumps. The integrated platform achieved a DNA yield of 2804.33 ± 15.31 ng/μL, a 5.9-fold increase over manual magnetic bead extraction. TaqMan quantitative PCR (qPCR) validation targeting the EHP SSU rRNA gene was developed. Using a standard curve spanning 101 to 107 plasmid copies (Ct = -3.611 log10 [copy] + 42.309, R2 = 0.998, amplification efficiency 89.2%), the on-chip MSPE achieved a validated analytical limit of detection (LOD) of 1 spore per reaction (100% detection rate, n = 21), whereas a commercial CTAB-based DNA extraction kit failed to achieve a validated LOD even at 10 spores (85.7%, 18/21). Nested PCR targeting the SWP gene was employed for field evaluation. A pilot study across two cohorts (N = 40) demonstrated consistent detection of confirmed EPH-positive cases; however, the small sample size precludes definitive diagnostic accuracy claims. With a total processing time under 30 min, this platform provides a high-efficiency extraction module. Future work will couple the device with isothermal amplification (e.g., LAMP or RPA) to realize a sample-to-answer system for resource-limited aquaculture.
