Passively driven microfluidic device with simple operation in the development of nanolitre droplet assay in nucleic acid detection

Abstract

Since nucleic acid amplification technology has become a vital tool for disease diagnosis, the development of precise applied nucleic acid detection technologies in point-of care testing (POCT) has become more significant. The microfluidic-based nucleic acid detection platform offers a great opportunity for on-site diagnosis efficiency, and the system is aimed at user-friendly access. Herein, we demonstrate a microfluidic system with simple operation that provides reliable nucleic acid results from 18 uniform droplets via LAMP detection. By using only micropipette regulation, users are able to control the nanoliter scale of the droplets in this valve-free and pump-free microfluidic (MF) chip. Based on the oil enclosure method and impermeable fabrication, we successfully preserved the reagent inside the microfluidic system, which significantly reduced the fluid loss and condensation. The relative standard deviation (RSD) of the fluorescence intensity between the droplets and during the heating process was < 5% and 2.0%, respectively. Additionally, for different nucleic acid detection methods, the MF-LAMP chip in this study showed good applicability to both genome detection and gene expression analysis.

Figure 1

Schematic illustration of the passive-driven MF-LAMP chip. (A) Photograph of the MF-LAMP chip with blue dye (bright field) and fluorescence dye (fluorescence image) to indicate the microfluidic network. Scale bars are 3 mm, (B) Bright field image of a single chamber. Scale bars are 0.5 mm. (C) Simple operation of bidirectional fluid regulation by using micropipettes integrated into the MF-LAMP device. Illustration of mechanism is visualized by PowerPoint. (D) The process of buffer exchanging and oil enclosed method through capillary channel. (E) Principle of the oil enclosed and impermeable layer that prevents LAMP reagent loss by liquid/gas diffusion through the inlet and PDMS. Illustration of mechanism is visualized by PowerPoint.

Figure 2

Fluid substitution performance of the MF-LAMP chip. (A,B) Schematic of the buffer inlet direction of the MF-LAMP chip. Illustration of mechanism is visualized by PowerPoint. (C) Illustration of the computational simulation domain in a single chamber with various capillary channel widths (10, 30 and 50 µm) visualized by COMSOL. (D) Comparison of the fluid substitution efficiency in the simulation result. (E) Time-sequence snapshots of the dynamics of dilution by water injection in the fluorescence droplet and the relative intensity variance (F) throughout the injection process.

Figure 3

Oil enclosure performance of the MF-LAMP chip with varied capillary channels. (A) Schematic of the oil inlet direction of the MF-LAMP chip. (B) Capillary force that stopped oil insertion into the reaction chamber. Illustration of mechanism is visualized by PowerPoint. (C) Computational simulation of the single chamber domain through the oil enclosure process with varied channel widths (10, 30 and 50 µm) and reagent volume loss. Illustration is visualized by COMSOL (D). (E) Actual oil enclosure process with blue dye used as the LAMP reagent for visualization; the reagent volume variation is plotted in (F) and shown as red dots.

Figure 4

Application of the MF-LAMP chip. (A) Different fabrication strategies of MF-LAMP with or without enclosed oil. The colored ink and fluorescence dye allow better observation of the variation of different permeable methods after the heating process. Illustration is visualized by PowerPoint. (B) Comparison of each method according to fluorescence variance between the initial status and the after heating status. (C) Analysis of the uniformity of the intensity in each reaction chamber with different fluorescence concentrations. (D) Amplification curves for detecting different concentrations of E. coli DNA (10³, 10², 10, and 1 pg µL⁻¹) generated by the processing of the fluorescence images. Illustration of mechanism is visualized by PowerPoint.