Self-digitization chip for quantitative detection of human papillomavirus gene using digital LAMP

Abstract

Digital nucleic acid amplification and detection methods provide excellent sensitivity and specificity and allow absolute quantification of target nucleic acids. Isothermal methods such as digital loop-mediated isothermal amplification (digital LAMP) have potential for use in rapid disease diagnosis in low-resource settings due to their speed and lack of thermal cycling. We previously developed a self-digitization (SD) chip, a simple microfluidics device that automatically digitizes a sample into an array of nanoliter wells, for use in digital LAMP. In this work, we improve the SD chip design to increase sample loading efficiency, speed, and completeness, and test a range of well volumes and numbers. We demonstrate the diagnostic capability of this platform by applying it to quantifying human papillomavirus 18 gene.

Figure 1.. The self-digitization (SD) chip.

(A) The SD chip consists of an array of channels and wells that spontaneously compartmentalizes aqueous samples into defined volumes for digital nucleic acid quantification assays. (B) The sample loading process is illustrated in single well schematics (top) and photographs (bottom). (i) The device is primed with an oil mixture to eliminate air. (ii) Aqueous sample is loaded, travels through channels, and expands into wells to lower the surface energy. Hydrophobic drainage channels assist sample filling by allowing oil to drain from the wells but are too small to allow aqueous sample to transfer. (iii) Additional oil is loaded and displaces aqueous solution from the channels but not from the wells, isolating the aqueous solution in compartments (iv). (C) Enhancements of the SD chip design include (a) using beveled well edges to reduce high-energy distortions of aqueous droplets; (b) using “Greek key” drainage channels with a height difference between the wells and drainage channels to improve oil drainage; (c) notches in the channels on both sides of the wells to facilitate oil breakoff during digitization; and (d) a notch in the channel opposite the well to direct aqueous solution into the well during filling.

Figure 2.

The SD chip design digitizes samples over a range of well volumes and numbers. Top row: photographs of device masters; bottom row: fluorescence images of digitized samples containing fluorescein (A shows full array, B-D are partial arrays to facilitate visualization). Four SD chips were designed with different well numbers and dimensions: (A) 640 wells (1000 μm long × 520 μm wide × 200 μm tall per well) with individual well volumes of ~100 nL and a total volume of >60 μL; (B) 1024 wells (400 μm (l) × 200 μm (w) × 100 μm (h) per well) with individual well volumes of ~7.5 nL and a total volume of ~8 μL; (C) 25,600 wells (160 μm (l) × 80 μm (w) × 80 μm (h) per well) with a well volume of ~1 nL and a total volume of >25 μL; D) 10,240 wells (30 μm (l) × 50 μm (w) × 35 μm (h) per well) with a well volume of ~50 pL and a total volume of ~0.5 μL..

Figure 3.

The completeness and uniformity of well filling were assessed using an SD chip with 1024 wells of 7.5 nL each. A-E) Fluorescence stereoscope images of five replicates of arrays loaded with fluorescein solution. F) The average normalized well volume for each replicate was calculated based on normalized integrated pixel intensities. The five replicates showed near complete fill fractions (95–97%) and high monodispersity (CV <1.4%).

Figure 4.

A) Three replicates of dLAMP HPV-18 assay. The HPV-18 concentration of the original sample was about 3.1×10⁴ molecules/mL. The arrays consisted of 1,536 wells with a well volume of 6.5 nL and a total volume of 10 μL. B) A plot of the average well intensities from images in (A). Red dots represent wells defined as positive (HPV-18 detected); blue dots represent wells defined as negative (no HPV-18). C) Absolute quantification results: the ratio of measured and expected HPV-18 concentrations for six devices loaded with different HPV concentrations. Dashes in the figure indicate 95% confidence intervals based on expected concentrations. For Devices 1–4, the ratio of measured to expected concentration fell within the 95% confidence interval. For Device 5, two of the three ratios fell within the 95% confidence interval. A negative control containing no template was also performed, which had only two false-positive wells over three replicates. Devices 7 tested the specificity of the assay. Control experiment for specificity was also performed containing HPV 16, 31, and 45 plasmids at 3.1×10⁴ molecules/mL each, and the expected concentration for the HPV-18 target is zero so the ratio is undefined. The three control replicates had zero, one, and three false-positive wells, an overall false-positive rate of ~0.1% (data not shown). Device 7 contained HPV 16, 18, 31, and 45 plasmids at 3.1×10⁴ molecules/mL each. One of three Device 7 arrays showed a ratio within the 95% confidence interval, but the average of the three also fell within the interval.