Lyophilization

An RPA nfo master mix for 24 reactions was prepared without adding enzymes, i.e. including rehydration buffer, primers and probes, and magnesium acetate. The master mix was vortexed thoroughly, then 8 μL were aliquoted into PCR tubes. Separately, 24 RPA nfo enzyme pellets were reconstituted in 1.2 mL of nuclease-free water and vortexed thoroughly. 10 μL of the reconstituted enzymes were aliquoted into 96 PCR tubes. Six milliliters of 0.5 U/μL ACP were prepared as previously described and aliquoted in 25 μL increments into individual PCR tubes. Master mix, enzyme, and ACP aliquots were frozen at −20 °C for a minimum of 2 hours, −80 °C for a minimum of 2 hours, and liquid nitrogen for 10 seconds prior to lyophilizing for a minimum of 24 hours (LabConco FreeZone 12, Kansas City, MO). As previously described, 40 μL of diluted gold nanoshells were deposited onto glass fiber pads and lyophilized for 24 hours without freezing prior to lyophilization. Lyophilized reagents were stored at −20 °C (amplification reagents, enzymes) or room temperature (glass fiber pads) in foil pouches with desiccant for up to one month.