Multiplexed Centrifugal Microfluidic System for Dynamic Solid-Phase Purification of Polynucleic Acids Direct from Buccal Swabs

Abstract

This report describes the development of a centrifugally controlled microfluidic dynamic solid-phase extraction (dSPE) platform to reliably obtain amplification-ready nucleic acids (NAs) directly from buccal swab cuttings. To our knowledge, this work represents the first centrifugal microdevice for comprehensive preparation of high-purity NAs from raw buccal swab samples. Direct-from-swab cellular lysis was integrated upstream of NA extraction, and automatable laser-controlled on-board microvalving strategies provided the strict spatiotemporal fluidic control required for practical point-of-need use. Solid-phase manipulation during extraction leveraged the application of a bidirectional rotating magnetic field to promote thorough interaction with the sample (e.g., NA capture). We illustrate the broad utility of this technology by establishing downstream compatibility of extracted nucleic acids with three noteworthy assays, namely, the polymerase chain reaction (PCR), reverse transcriptase PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). The PCR-readiness of the extracted DNA was confirmed by generating short tandem repeat (STR) profiles following multiplexed amplification. With no changes to assay workflow, viral RNA was successfully extracted from contrived (spiked) SARS-CoV-2 swab samples, confirmed by RT-qPCR. Finally, we demonstrate the compatibility of the extracted DNA with LAMPa technique well suited for point-of-need genetic analysis due to minimal hardware requirements and compatibility with colorimetric readout. We describe an automatable, portable microfluidic platform for the nucleic acid preparation device that could permit practical, in situ use by nontechnical personnel.

Centrifugal Microfluidic Method for Enrichment and Enzymatic Extraction of Severe Acute Respiratory Syndrome Coronavirus 2 RNA

“…Within a single domain, NP enrichment and enzymatic lysis occur in the centrally located magnetic manipulation chamber. This “concave-shaped” 18 chamber features a distal vertex, designed to retain magnetic NPs during supernatant removal and, ultimately, RNA elution. The liquid sample, suspended NPs, and the enzymatic extraction cocktail were loaded into the sample input, magnetic manipulation, and extraction reagents chambers, respectively ( Figure 3 D).…”

“…Valve opening, 21 channel closures, 19 rotationally driving flow, and magnetic mixing 18 were enabled by in-house mechatronic systems regulated by 8-core microcontrollers (Propeller P8X32A-M44; Propeller, Inc., Rockland, CA) and custom programs written in Spin , Propeller’s coding language, run from a laptop computer. On-disc heating was facilitated by a clamped, dual-Peltier system.…”

“… 16 , 17 Notably, the NPs are functionalized with iron oxide, allowing for efficient mixing in the system via application of an external, bidirectional rotating magnetic field (bRMF) that facilitates NP “sweeping”. 18 Furthermore, active, laser-actuated valving permits both valve opening and channel closures to enable reliable implementation of numerous sequential unit operations. 19 Here, we demonstrate the compatibility of our optimized method for rapid SARS-CoV-2 sample preparation with full-process controls and clinical samples in multiple, clinically relevant matrices.…”

Nucleic acid extraction without electrical equipment via magnetic nanoparticles in Pasteur pipettes for pathogen detection

“…Lysis buffer and washing buffer were prepared by referencing the reports of Dignan et al, Liu et al and Wang et al with some modifications [ [25] , [26] , [27] ]. Specifically, 200 μL samples and 30 μL MNPs suspension were added into a mixture composed of 300 μL lysis buffer (50 mM Tris, 10 mM EDTA, 0.1 M NaCl, 4 M guanidine isothiocyanate, 0.1% SDS [w/v], 2 mg/mL proteinase K, 5% Tween-20 [v/v], and 3% TritonX-100 [v/v], pH 8.0) and 300 μL isopropanol in a 1.5 mL centrifuge tube.…”

A Comparative Evaluation of Indirect Sequence-Specific Magnetoextraction-aided Fluorescence and Electrochemical LAMP with SARS-CoV-2 Nucleic Acid as the Analyte

“…The rest of the methods predominantly are either whole nucleic acid extraction or immunomagnetic capture followed by lysis. The former also included all magneto-extraction associated SARS-CoV-2 detection techniques using LAMP (entries 7, 10, 14, 24, 35, 48, Table S1 25,[33][34][35][36][37] ). Surprisingly, none of these studies listed in Table S1 utilized electrochemical readout despite its promising potential in limited resource detection.…”