L7012

For the Staphylococcus aureus enrichment test case, we used a Zymo bacterial sample kit (D6310), a premixed sample of microbes containing eight bacterial species (three Gram-negative and five Gram-positive) and two species of yeast. We verified the ratio of microbes by sequencing. This sample was washed three times by centrifugation and resuspended with 1 mL of phosphate-buffered saline (PBS).
Bacterial abundance was determined using a bacterial live/dead assay (L7012; Thermo Fisher Scientific) and hemacytometer (DHC-N21; Bulldog Bio) before proceeding to the encapsulation step. For the Antarctic sample (a gift from Roger Summons), a small portion of the freeze-dried sample was immersed in 1 mL of PBS in a 1.5-mL Eppendorf tube. The tube was then taped onto a vortex mixer and vortexed for 10 min at high speed to resuspend single bacteria.
The suspension was then filtered using a 40-μm strainer (352340; Falcon) to remove large debris and a 5-μm filter (SLSV025LS; Millex-SV) to remove any particles that could potentially clog the microfluidic channel (while allowing bacteria to pass through). The sample was then washed three times with centrifugation and resuspended in 1 mL of PBS to remove any free-floating DNA.
Finally, bacterial abundance was determined using a bacterial live/dead assay (L7012; Thermo Fisher Scientific) and hemacytometer (DHC-N21; Bulldog-Bio).

Viability of the yeast cells on the electrode was examined by analyzing the membrane permeability changes with a live/dead backlight bacterial viability kit for microscopy and quantitative assays according to the manufacturer's recommendations (L7012, Molecular Probes, Eugene, OR, USA). To obtain dead yeast cells to act as a benchmark, we cultured yeast cells with 1% (v/v) Tween 20 (Wako, Osaka, Japan) containing PBS(−) for 60 min at 60ºC. After constant potential application, the yeast cells on the patterned working electrode were washed with 25 ml of PBS(−) and stained with the live/dead backlight bacterial viability kit for 20 min at RT (Fig. 1). After incubation for 20 min, the patterned working electrode was again washed with 25 ml of PBS(−) and observed using an epifluorescence microscope system (BX51, Olympus, Tokyo, Japan) connected to a digital camera (DP72, Olympus) and image analysis system software (DP2-BSW, Olympus).
For the measurement of yeast cell density on the patterned working electrode, the numbers of yeast cells on the electrodes were counted in random areas of 100 × 100 μm² after potential application. Measurement of yeast cell density was repeated eight times, and data were expressed as the mean of two independent experiments.

C-terminal amidated PuroA (FPVTWRWWKWWKG-NH₂) and FITC-labelled PuroA (FITC-FPVTWRWWKWWKG-NH₂) were synthesized with >95% purity by solid-phase methods using N-(9-fluorenyl) methoxycarbonyl (Fmoc) chemistry at Biomatik Crop (Ontario, CA). Peptides used without further purification. Peptide solutions were made in sterile phosphate-buffered saline (PBS; 140 mM NaCl, 2.5 mM KCl, 1.6 mM KH₂PO₄, 15 mM Na₂HPO₄, pH 7.4). A 20 mM solution (in DMSO) of propidium iodide and A 5 mM solution (in DMSO) of SYTO 85 Orange fluorescent nucleic acid stain were purchased from Molecular Probes (L7012 and S11366, respectively). All aqueous solutions were made in sterile, ultrapure (18 MΩ) water which was quartz-distilled and deionised in a Milli-Q^TM system (Millipore, Bedford, MA, USA).