The device was designed to be injection-molded for mass production. For fast prototyping, we fabricated mockup devices via mechanical machining. Polycarbonate, which is one of the plastic materials for injection-molding, was used as a structural material and machined via micro-milling (100 μm micro-end mill). The sample processing kit is divided into four parts: two inner halves for sample processing and two outer halves for sample storage. The assembled kit (24 × 60 × 65 mm3) has two chambers, one for the oral swab and the other for pumping. Each chamber is fitted with a plunger that can be manually actuated through twisting motions. Other key design features are: (i) the swab chamber includes an inline filter (pore size, 0.45 μm; HAWP02500, MilliporeSigma) to remove debris from natural oral fluid; (ii) the oral fluid reservoir has overflow openings to collect a fixed volume of oral fluid (20 μL). The collected oral fluid then mixes with AuNPAb (50 μL) that were preloaded into the device. AuNPAb are retained under the oral fluid reservoir with off-axis flow alignment with the reservoir outlet (see Fig. S2 for details); (iii) the fluidic channel has a beehive-like expansion structure to enhance the mixing efficiency between oral fluid and AuNPAb (57 (link)–59 (link)); and (iv) the two processing kit outlets line up with the sensor cartridge inlets for seamless sample delivery.
Hawp02500
Manufactured by Merck Group
Sourced in United States
HAWP02500 is a laboratory instrument designed for general analytical and research purposes. It is a versatile piece of equipment that can be used in various scientific applications. The core function of this product is to perform precise measurements and data analysis as required by the user's specific needs. No further details on the intended use or capabilities of this equipment are provided.
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Lab products found in correlation
Scintillation fluid, by Beckman Coulter (2 mentions)
35sγgtp, by PerkinElmer (2 mentions)
Xx2702550 vacuum manifold, by Merck Group (2 mentions)
Triathler liquid scintillation counter, by Hidex (2 mentions)
D1408, by Merck Group (2 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Gvwp02500, by Merck Group (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
14 protocols using hawp02500
1
Injection-Molded Oral Fluid Sampling Device
2
Quantitative RNA-Protein Binding Assay
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Affinity constants (KD values) were determined by a radiolabeled binding assay. Approximately 15,000–20,000 CPM of labeled and refolded RNA was incubated with varying concentrations of RT (0.1–1000 nM or without RT to determine background binding) in binding buffer (50 mM Tris-HCl [pH 7.5], 140 mM KCl, 1 mM MgCl2, and 0.1 μg/mL BSA) and allowed to equilibrate on ice for 15 min. Assembled RNA:RT complexes were then partitioned from unbound RNA by passing samples over a nitrocellulose filter (HAWP02500; Millipore Sigma) under vacuum (XX2702550; Millipore Sigma) and immediately washing with 500 μL binding buffer. Radioactivity retained on filters was counted by adding 4 mL of scintillation fluid to filters placed inside of scintillation vials and counted using a liquid scintillation counter. Fraction of RNA bound was calculated by determining the fraction of radioactivity bound and were fit to a one-site, specific binding curve using Prism GraphPad 6.2. Replicates of binding assays were performed using recently prepared single batches of each protein and aptamer for all KD measurements involving that protein or aptamer, to avoid drift of signal from aging samples or from batch-to-batch variation.
3
Contact Angle Measurements of S. gordonii
4
Archiving and Preserving Seawater Samples
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Each sample collected for archiving was filtered through a puck containing a 0.22 µm duropore filter (GVWP02500, Millipore, USA) on top of a 0.45 µm (HAWP02500, Millipore, USA) filter. The purpose of the 0.45 µm filter was to ensure that the 0.22 µm remain soaked with RNAlater for appropriate DNA preservation. Immediately after filtration archival samples were evacuated of seawater and preserved with 4 mL of RNAlater (Life Technologies, USA) for 2 min each, before RNAlater was evacuated, and the preserved sample stored onboard the ESP at RT (17–18 °C). After instrument recovery, filters were transferred to a freezer and stored at − 20 °C until DNA extraction. Laboratory based DNA extraction and analysis mimicked those on the ESP, thus using a modified lysis and purification protocol using the DNeasy Blood and Tissue kit (Qiagen, USA) column. For DNA purification and final elution equivalent volumes to the ESP were used. For subsequent laboratory analyses of the archived DNA, all qPCR reactions were scaled down to 10 µL reactions to allow analysis in triplicates and analyzed on the StepOnePlus under thermocycling conditions mimicking the ESP. The triplicate 10 µL reaction setup utilized for the archival samples enables equal detection and quantification ability as the singlicate 30 µL in situ reaction setup by analyzing a total of 6 µL DNA in both procedures.
5
Streptomyces Spore Preservation and Growth Curves
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Streptomyces spores were obtained from MS agar plates and preserved in 20% glycerol at −20°C (Kieser et al., 2000 ). Liquid cultures were grown in LB + 25% sucrose or 2xYT (Kieser et al., 2000 ), without sucrose. Growth curves were performed in 2xYT or YEME media (Kieser et al., 2000 ), using freshly harvested spores to give an initial OD450 = 0.01. Samples (10 mL) were taken at the indicated times and filtered through 0.45 μm cellulose acetate filters (Millipore, HAWP02500); the mycelium was then dried at 100°C for dry weight determination. Total actinorhodin was determined by lysing 1 mL aliquots of the culture with 1 M KOH, which were then centrifuged to remove debris and measuring OD640 (Kieser et al., 2000 ).
6
Rab33b Ubiquitination and GTP Incorporation
7
Rapid Dilution-Filtration Assay for Cl-36 Uptake
8
Rapid Dilution-Filtration Assay for Cl-36 Uptake
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Rapid dilution-filtration assays were performed as described previously.26 (link) MG1655 E. coli were grown to stationary phase as described above for the solute distribution measurements. An aliquot equivalent to 2 mL of OD600 = 2.0 was thrice washed and resuspended in 2 mL 10 mM HCl, pH 2, 150 mM KCl, 10 mM glucose and 1 mM glutamic acid. 36Cl was added at a concentration where 100 uL of the suspension contained approximately 80,000 counts per minute. After incubating for 20 minutes, 100 uL of the suspension was diluted into 10 mL unlabeled 10 mM HCl, pH 2, 150 mM KCl, 10 mM glucose and 1 mM glutamic acid and the solution was immediately vacuum-filtered through a 2.5 cm 0.45 um Nitrocellulose membrane (Millipore cat. no. HAWP02500) using a Millipore XX2702550 vacuum manifold. The time between dilution and complete filtration was about 15 seconds. The filter was then resuspended in 10 mL scintillation fluid and the radioactivity was assayed in a Hidex Triathler liquid scintillation counter. Three replicates were performed. To determine the amount of 36Cl from residual liquid retained on the filter, the above dilution-filtration procedure was performed on a 2 mL sample of buffer containing 80,000 cpm 36Cl per 100 uL without E. coli.
9
Quantification of Glycerol 3-Phosphate
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G3P pools reported in Fig. 4d were measured as follows. The culture of NQ1187 was grown to OD600 = 0.5, and the cells were harvested by filtration of 2.5 ml culture through the membrane filter (25 mm-disc with 0.45 μm pore size, HAWP02500; Millipore) pre-wetted with warmed culture medium, and washed by 2.5 ml warmed culture medium. The filter was quickly immersed in 4 ml of extraction solution (40% (v/v) methanol, 40% (v/v) acetonitrile, and 20% (v/v) water) precooled at −20°C, and incubated at −20°C for 2 h. The extract was dried in a vacuum concentrator and stored at −80 °C. Immediately prior to the assay, the samples were dissolved in 170 μl phosphate-buffered saline (D1408; Sigma-Aldrich). G3P was assayed enzymatically using a commercially available kit (Amplite Fluorimetric Glycerol 3-Phosphate Assay Kit, 13827; AAT Bioquest).
10
Antibiotic Resistance Gene Transfer
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Transfer of antibiotic-resistant genes from LAB isolates to pathogenic microorganisms was examined by the filter mating method [13 (link)]. For this, LAB isolate (donor) and intestinal pathogen (recipient) were grown separately in a non-selective medium up to the mid-exponential phase of growth (~ 4h). The donor cells and the recipient cells were mixed in 1:1 ratio, and the mixture was filtered through a sterile composite cellulose ester filter (0.45μm, HAWP-02500, Millipore) using a swinex filter holder (SX 02500, Millipore). After filtration, sterile peptone physiological saline solution (PPS- 8.5gm/L NaCl and1gm/L peptone) was passed through the filters to trap the cells more tightly into the membrane. The filters were placed on a non-selective agar medium and incubated at recipient bacterial growth conditions. After incubation, filters were washed with 2ml PPS solution, filtrates (mating mixture) were spread on selective antibiotic agar medium plates and incubated at 37°C for 24–48 h, to screen for antibiotic-resistant transconjugants.
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