Warmstart lamp master mix

LAMP assay solutions were prepared by aliquoting 12.5 µL of WarmStart LAMP Master Mix (New England Biolabs, Whitby, ON, Canada), 0.5 µL of fluorescent dye (New England Biolabs, Whitby, ON, Canada), 2.5 µL of novel LAMP primers and probe mixture (100 nL FIP, 100 nL BIP, 12.5 nL F3, and 12.5 nL B3 in 2.275 µL nuclease-free water), and 7 µL of nuclease free water. 2.5 µL of SARS-CoV-2 RNA from extracted clinical and environmental samples was added to the mix, 12.5 µL of the 25 µL mix was aliquoted into the oil pre-filled microfluidic chip, and the assay droplet was positioned at the T-junction of the microfluidic chip.

We performed bulk RT-LAMP and DNA logic assays on purified RNAs, cells and mixtures of cells in triplicate using a Bio Rad CFX Connect quantitative PCR (qPCR) machine. We incubated the reactions at 65°C and monitored FAM, HEX and/or SYBR fluorescence channels. Each reaction comprised a total volume of 10 μl, consisting of 1.6 μM each FIP/BIP primer, 0.2 μM each F3/B3 Primer, 0.4 μM each LoopF/B Primer, 1× WarmStart LAMP Master Mix (New England Biolabs) and 0.5 U/μl SUPERase•In™ RNase Inhibitor (Invitrogen). We added DNA complexes at varying concentrations given in Supplementary Table S2. LAMP primer and logic gate sequences are shown in Supplementary Table S1. In reactions without any DNA complexes, we included LAMP Fluorescent Dye (New England Biolabs) as a general LAMP indicator. For experiments performed on purified RNAs, we added in vitro transcribed KRT19, VIM and/or PTPRC RNAs at the concentrations shown in Supplementary Table S2. For experiments performed on cells, we also included 2.5% Tween-20 (Sigma Aldrich) in the reaction buffer to act as a lysis reagent. We then added intact, unstained cells immediately before starting an experiment at a final concentration of 50 cells/μl per cell type.

PEG hydrogel
monomers included 4-arm PEG-acrylate [molecular weight (MW) of 10 000,
Laysan Bio, Arab, AL, USA] and thiol-PEG-thiol (MW of 3400; Laysan
Bio), with acrylate and thiol mixed at a molar ratio of 1:1 for cross-linking.
For sol–gel transition time characterization, 7.5 w/v% and
10 w/v% PEG hydrogel were respectively tested in PCR mix, LAMP mix,
and culture media mix. PEG monomers were weighed to make 10×
monomer solutions for PEG-acrylate and PEG-thiol separately. The weighed
monomers were then dissolved either in water (Molecular Biology grade
Water, Corning, Acton, MA, USA) for PCR and LAMP mix, or in TSB (BD
Bacto Tryptic Soy Broth, Becton Dickinson and Company, Franklin Lakes,
NJ, USA) for culture media mix. In addition to 2 μL of each
10× PEG monomer solution, for each 20 μL of reaction mix,
the PCR mix contained 10 μL of ddPCR Supermix for Probes (BioRad,
Hercules, CA, USA) and 6 μL of water; the LAMP mix contained
10 μL of 2× WarmStart LAMP Mastermix (New England Biolabs,
Ipswich, MA, USA) and 6 μL of water; and the culture media mix
contained 16 μL of TSB. The reaction mix was briefly vortexed.
The sol–gel transition was considered started when lifting
the pipet tip could draw filaments out of the reaction mix, and the
transition was considered ended when the reaction mix formed a gelatinous
lump.