Fig 1.
Workflow of sample loading and chip packaging.
(a) The sample loading instrument. (b) Scraping reagents into picoliter wells and overlaying the chip with excess mineral oil. (c—d) Filling the copper chamber with mineral oil prior to transferring and packaging the finished PWA chip. (e—f) Fixing a glass cover-plate on the copper chamber with screws.
Fig 2.
(a) Isothermal incubation setup and wide-field fluorescence imaging setup. (b) Scanning electron microscope (SEM) image of the picoliter well array. Scale bar represents 300 μm. (c) Passivating the PWA chip surface by covalently coupling methoxy-PEG-silane.
Fig 3.
Digital RPA on the non-silanization and silanization chip.
Real-time images of RPA amplification at 5 min and 10 min are shown. For a gDNA sample concentration of 0.53 pg μL-1, there are 1.2 copies of gDNA per 100 wells, on average. Scale bar represents 1 mm. (a) Many clusters of positive RPA wells appeared gradually from 5 min to 10 min, with cross-contamination on the non-silanization chip. (b) Positive RPA wells amplified independently from 5 min to 10 min, without cross-contamination on the silanized chip.
Fig 4.
Real-time imaging of RPA amplification for 0–20 min.
Scale bar represents 2 mm.
Fig 5.
Digital RPA on the PWA chip for different concentrations of Listeria monocytogenes gDNA.
(a—e) Digital RPA on the PWA chip with serial dilutions of target DNA template ranging from 9 × 10-1 to 4 × 10-3 expected copies per well (cpw). (f) Control, no wells showed positive signals when no target DNA was loaded.
Fig 6.
Quantitative results of digital RPA on the PWA chip.
The measured copies per well (cpw) was highly concordant with the expected cpw, with an average error rate of less than 11% (N = 15). Error bars represent standard deviations.