Fluorinert FC40 (3M, Sigma Aldrich, UK) was used for the formation of the two‐phase systems to be used as cell culture substrates. The main physical properties of Fluorinert FC40 are listed in Table 1 . Initially, the perfluorocarbon was sterilised by autoclaving at 121°C and 2 atm for 15 min. After use, the perfluorocarbon was recovered, recycled and re‐sterilised by vacuum filtration through a 0.2 µm filter unit (Millipore, UK). For all experiments, ultralow attachment 6 and 24‐well plates (Corning, UK) were employed to ensure cell attachment was only achieved on the tested substrates and not the plastic. For the preparation of the two‐phase systems, the perfluorocarbon FC40 was first added to the well plate, followed by the gentle addition of the growth medium so it formed a layer on top of the perfluorocarbon. A 1:2 ratio of perfluorocarbon to growth medium was used for all experiments. For example, in the 24‐well plates, 1 mL of perfluorocarbon was topped up with 2 mL of growth medium to ensure the formation of a flat interface.
Fluorinert fc 40
Manufactured by 3M
Sourced in United States
Fluorinert FC-40 is a clear, colorless, and odorless liquid manufactured by 3M. It is an inert, fluorinated liquid with a high boiling point and low surface tension. Fluorinert FC-40 is primarily used as an electronic coolant and in other industrial applications where a thermally and chemically stable liquid is required.
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Lab products found in correlation
Az1505 photoresist, by MicroChemicals (2 mentions)
Pb buffer, by Qiagen (1 mentions)
Koptec 200 proof ethanol, by Decon Labs (1 mentions)
Pe buffer, by Qiagen (1 mentions)
Zymo viral dna rna buffer, by Qiagen (1 mentions)
L arabinose, by Thermo Fisher Scientific (1 mentions)
Dynabeads m 280 tosylactivated, by Thermo Fisher Scientific (1 mentions)
Tween 40, by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
Tween 60, by Merck Group (1 mentions)
Emccd ixonem du 897 camera, by Oxford Instruments (1 mentions)
Origin 8, by OriginLab (1 mentions)
Ix81 inverted microscope, by Olympus (1 mentions)
Teflon af, by DuPont (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Rompun, by Bayer (1 mentions)
Sodium borohydride solution, by Merck Group (1 mentions)
Epichlorohydrin ech, by Merck Group (1 mentions)
2 propanol ipa, by Merck Group (1 mentions)
4 mu phosphate 4 mup, by Merck Group (1 mentions)
9 protocols using fluorinert fc 40
1
Fabrication of Two-Phase Cell Culture Substrates
2
Optimizing LAMP and qPCR Reactions
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For studying kit buffer inhibitors, LAMP and qPCR reactions were spiked to 5 × 104 cp/rxn λ phage DNA (NEB) and supplemented with half-log dilutions of either Koptec 200-proof ethanol (V1001, Decon Labs, King of Prussia, PA, USA), Viral RNA Wash Buffer 1x (R1034-2-48, Zymo Research, Tustin, CA, USA), Buffer PE (19065, Qiagen, Germantown, MD, USA), Zymo DNA/RNA Shield 1x (R1200-125), Zymo Viral DNA/RNA Buffer (D7020-1-100), or Qiagen Buffer PB (19066) to the appropriate final concentration. For selecting the optimal TPW, LAMP and qPCR reactions were spiked with 1 µL of 5 × 104 cp/µL λ phage DNA, diluted to 10 µL, and an additional 1 µL was added of either nuclease-free water, 200 proof ethanol, isopropanol (BP2618-500, Thermo Fisher Scientific, Waltham, MA, USA), 1-butanol (3000-04, Mallinckrodt Chemicals), isopentanol (2992-04, Mallinckrodt Chemicals), 1-hexanol (H13303–100 mL, MilliporeSigma, St. Louis, MO, USA), 1-heptanol (H2805-250 mL, MilliporeSigma), 1-octanol (SHBH2844V, MilliporeSigma), 1-nonanol (131210–100 mL, MilliporeSigma), 1-decanol (2397563–50 g, MilliporeSigma), 1-undecanol (MKCG3271, MilliporeSigma), 2-dodecanol (D221503-5G, MilliporeSigma), 5 cSt silicone oil (317667-250 mL, MilliporeSigma), or Fluorinert FC-40 (ZF-0002-1308-0, 3 M, St. Paul, MN, USA).
3
Fabrication of Microfluidic Biosensor
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LB, phosphate buffered saline (PBS), sodium chloride, Tween 40, Tween 60, and Tween 80 were purchased from Sigma Aldrich (Oakville, ON, USA). Fluorinert FC-40 was purchased from 3M (St. Paul, MN, USA) and chemicals for photolithography were supplied by Rohm and Haas (Marlborough, MN, USA). Fluoroalkylsilane Dynasylan F 8263 was supplied by Evonik (Essen, Germany). AZ1505 photoresist and Teflon-AFR were procured from Microchemicals GmbH (Ulm, Germany) and DuPont (Wilmington, DE, USA), respectively. Parylene-C dimer and Silane A174 were purchased from Plasma Parylene Coating Services (Rosenheim, Germany) and superparamagnetic Dynabeads M-280 Tosylactivated were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Ampicillin and isopropyl β-D-1-thiogalactopyranoside (IPTG) were procured from Applichem, Darmstadt, Germany, and L-Arabinose from Acros Organics. Mouse monoclonal anti-Salmonella typhimurium LPS antibody (ab8274) was obtained from Abcam (Cambridge, UK).
4
Microfluidic Synthesis of Hydrogel Microparticles
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To generate droplets, a microfluidic water-in-oil segmentation system was utilized. In a typical experimental configuration, the water phase consisted of an aqueous dispersion containing 1% (w/v) thiolated sodium alginate (SH-SA), 3.33% (w/v) vinyl-terminated hyperbranched (polyethylene glycol) diacrylate (HB-PEGDA), and 3.52 × 109 infectious units per milliliter (IFU/mL) of adenovirus. The oil phase, on the other hand, consisted of 3M™ Fluorinert™ FC-40 supplemented with 2 wt% Pico-SurfTM. The resulting droplets were collected in an Eppendorf tube and subsequently transferred to a drying oven, where the HMPs were cured at a temperature of 37 °C for a minimum of 1 h. To complete the process, the HMPs underwent purification by introducing Novec 7500 oil containing 20% v/v 1H, 1H, 2H, 2H-perfluoro-1-octanol (PFO) to eliminate any remaining surfactants. Following this, the HMPs were allowed to swell and equilibrate with buffer for a minimum of 2 h at 37 °C prior to their utilization. Fully swollen and equilibrated building block HMPs were pelleted at 8000 rpm for five minutes, and the excess buffer was removed to give the HMPs. All the catalog and company details of the materials used in this study are shown in Table S1 .
5
Microfluidic Encapsulation of Microalgae in Microdroplets
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A microfluidic device was used for encapsulation of microalgal cells into microdroplets. A suspension of cells in f/2 growth medium was injected as an aqueous phase in a flow-focusing microfluidic device of dimensions 25 μm × 50 μm (width × depth). The aqueous cell suspension flowed perpendicularly to two streams of fluorinated carrier oil Fluorinert™ FC-40 (3 M, United States) containing 2.5 wt% surfactant PicoSurf 1 (Sphere Fluidics, United Kingdom) (SI Fig. 1 A). The oil streams enveloped microdroplets that budded off from the aqueous stream and flowed away from the flow-focusing junction. The size of the microdroplets was tuned by changing the flow rate of the aqueous cell suspension or fluorinated carrier oil. The microdroplets were collected and stored in a 1 mL plastic syringe over a period of ~7 days to investigate their growth (SI Fig. 1 B). To monitor the growth of P. tricornutum and N. gaditana in microdroplets, microscope images were captured with an EMCCD iXonEM+ DU 897 camera (Andor Technology, United Kingdom) coupled with an IX 81 inverted microscope (Olympus, Japan). The number of cells in microdroplets was determined by counting cells using images taken in technical triplicate. The statistical analyses were performed with Origin 8.0 (OriginLab Co., USA), and the data were expressed as mean ± SD (standard deviation) of three replicates of counting.
6
Yeast Strain Culturing with Specialized Reagents
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Saccharomyces cerevisiae strain BY4741 was used in all experiments. Propidium iodide (PI; 540/608 nm) was supplied by Sigma Aldrich (Saint Louis, MO, USA). Amphotericin B was purchased from Sigma Aldrich (Saint Louis, MO, USA). Fluorinert FC-40 was purchased from 3M (St. Paul, MN, USA). Chemicals for photolithography, including S1818 and 351developer, were supplied by Rohm and Haas (Marlborough, MN, USA). AZ1505 photoresist was purchased from Microchemicals GmbH (Ulm, Germany). Parylene-C dimer and Silane A174 were purchased from Plasma Parylene Coating Services (Rosenheim, Germany). Teflon-AF® was obtained from Dupont (Wilmington, DE, USA).
7
Ex vivo MRI of Fixated Brain
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Fourteen months-old animals, both treated and control, were sacrificed under general anaesthesia (ketamine (100 mg/ml) (Imalgene 1000 ® , Merial) / xylazine (2%) (Rompun, Bayer)). Subsequently, they underwent a perfusion with a fixative solution of 10% formalin (37-40% formaldehyde (Panreac) in a phosphate buffer). After the fixation, the whole brain of each animal was removed, keeping the tissue at 4°C in a solution of 10% formalin until image acquisition.
The MRI scanner used ex vivo was the same as the one used to obtain images in vivo but with a different gradient and birdcage systems: gradient system of 6 cm with a maximum gradient strength of 450 mT/m, a rise time of 80 microseconds and a volume birdcage of 3 cm (Bruker). To carry out the MRI scanner, the brains were placed in Fluorinert FC-40 (3M, Minnesota, USA). T2W images were acquired using a 3D sequence FSE with the following parameters: TR = 1.6395 s; Effective ET = 72 ms; FOV = 2.5 x 2.5 x 2.5 cm 3 , NA = 1. The dimensions of the matrix acquisition and reconstruction were 256 x 192 x 64 and 256 x 256 x 64, respectively.
The MRI scanner used ex vivo was the same as the one used to obtain images in vivo but with a different gradient and birdcage systems: gradient system of 6 cm with a maximum gradient strength of 450 mT/m, a rise time of 80 microseconds and a volume birdcage of 3 cm (Bruker). To carry out the MRI scanner, the brains were placed in Fluorinert FC-40 (3M, Minnesota, USA). T2W images were acquired using a 3D sequence FSE with the following parameters: TR = 1.6395 s; Effective ET = 72 ms; FOV = 2.5 x 2.5 x 2.5 cm 3 , NA = 1. The dimensions of the matrix acquisition and reconstruction were 256 x 192 x 64 and 256 x 256 x 64, respectively.
8
Functionalization of Agarose Beads
9
Influenza A Virus Hemagglutinin Assay
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The influenza A virus [A/Puerto Rico/8/1934IJH1N1)] was prepared as previously reported. 45 2′-(4-Methylumbelliferyl)-α-D-Nacetylneuraminic acid (MUNANA) (Life Technologies, Carlsbad, CA, USA), Fluorinert-FC40 (3M), CYTOP (Asahi-glass, Tokyo, Japan), a highly active alkaline phosphatase recombinant (ALP, Roche, Switzerland), Fomblin Y-LVAC25/6 (Solvay, Brussels, Belgium), 4-MU phosphate (4MUP, Sigma-Aldrich, St. Louis, MI, USA), and RIDT (Quicknavi Flu; Denka Seiken, Tokyo, Japan) were purchased from the respective suppliers.
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