Flow-through Capture and in Situ Amplification Can Enable Rapid Detection of a Few Single Molecules of Nucleic Acids from Several Milliliters of Solution

Abstract

Detecting nucleic acids (NAs) at zeptomolar concentrations (few molecules per milliliter) currently requires expensive equipment and lengthy processing times to isolate and concentrate the NAs into a volume that is amenable to amplification processes, such as PCR or LAMP. Shortening the time required to concentrate NAs and integrating this procedure with amplification on-device would be invaluable to a number of analytical fields, including environmental monitoring and clinical diagnostics. Microfluidic point-of-care (POC) devices have been designed to address these needs, but they are not able to detect NAs present in zeptomolar concentrations in short time frames because they require slow flow rates and/or they are unable to handle milliliter-scale volumes. In this paper, we theoretically and experimentally investigate a flow-through capture membrane that solves this problem by capturing NAs with high sensitivity in a short time period, followed by direct detection via amplification. Theoretical predictions guided the choice of physical parameters for a chitosan-coated nylon membrane; these predictions can also be applied generally to other capture situations with different requirements. The membrane is also compatible with in situ amplification, which, by eliminating an elution step enables high sensitivity and will facilitate integration of this method into sample-to-answer detection devices. We tested a wide range of combinations of sample volumes and concentrations of DNA molecules using a capture membrane with a 2 mm radius. We show that for nucleic acid detection, this approach can concentrate and detect as few as ∼10 molecules of DNA with flow rates as high as 1 mL/min, handling samples as large as 50 mL. In a specific example, this method reliably concentrated and detected ∼25 molecules of DNA from 50 mL of sample.

Self-partitioning SlipChip for slip-induced droplet formation and human papillomavirus viral load quantification with digital LAMP

“…(Kang et al, 2014;Zhang et al, 2015b) Therefore, the sp-SlipChip can potentially enable digital nucleic acid quantification and cell analysis directly from physiologically relevant body fluids. Furthermore, the sp-SlipChip might be seamlessly integrated with on-chip sample preparation methods (Mahalanabis et al, 2009;Schlappi et al, 2016) as well as with easy readout methods (Rodriguez-Manzano et al, 2016) for sample-in-answer-out total analysis in resource-limited settings.…”

Unveiling massive numbers of cancer-related urinary-microRNA candidates via nanowires

“…Compared to the charge-based isolation approach for EVs ( 49 ), by which protamine precipitation of EVs is performed using protamine/polyethylene glycol in an overnight incubation at 4°C (similar to ExoQuick), we can say that our device has an advantage of providing 40-min in situ extraction of urine EV–encapsulated miRNAs (collection, 20 min; extraction, 20 min) at room temperature. Moreover, compared to the charge-based isolation approach for free nucleic acid ( 50 ), which uses chitosan polymer with amine groups that provide a positively charged surface only below pH 6.3, we can say that our device has an advantage of an assured positively charged surface at pH 6 to 8 of urine. Although the exact collection mechanism of EVs by nanowires in the present work must be rather complex, our findings indicated that the mechanically stable anchoring of nanowires anchored into PDMS during lysis buffer flow and the electrostatic collection of EVs (and, moreover, EV-free miRNAs) onto ZnO nanowires were two key mechanisms, and they will lead to realization of early disease diagnoses and timely medical checkups based on urine miRNA analysis.…”

Slip-driven microfluidic devices for nucleic acid analysis

“…To ensure the high quality of nucleic acid analysis performance on slip-driven microfluidic devices, the sample preparation module needs to be effective and efficient. There have been several microfluidic approaches for on-chip nucleic acid sample preparation, 67,68 and it will be interesting to see if these existing methods or any new methods can be seamlessly integrated with the slip-driven microfluidic devices.…”