Preparation of 3% w/w Kolliphor EL/ 2% w/w Pluronic P105 surfactant solution is reported previously (Patel S. K. et al. 2013 ). Emulsification protocol developed previously (Janjic et al. 2008 (link); O’Hanlon et al. 2012 (link)) was partially modified. First, surfactant solution was added to the oil(s). If present, solubilizer or PFPE-tyramide dissolved in transcutol (at 50 mg/mL) was added to the mixture. Then, coarse emulsions were produced with an analog vortex mixer (VWR, Radnor, PA) on high for 30 seconds. Triphasic emulsions were sonicated for 30 seconds at 29% amplitude with Model 500 Ultrasonic Dismembrator (FisherScientific, Pittsburgh, PA). Microfluidization was performed on all coarse emulsions by Microfluidizer M110S (Microfluidics Corp., Westwood, MA) at 15,000 psi liquid pressure for 20 pulses over ice cold interaction chamber. Emulsions were stored at 4 °C in glass vials. This method was used to produce all presented formulations. Microfluidization processing parameters were kept constant throughout the study.
Analog vortex mixer
Manufactured by Avantor
Sourced in United States
The Analog Vortex Mixer is a laboratory equipment used to create a vortex motion in liquid samples. It provides a simple and effective way to mix liquids in test tubes or other containers.
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Lab products found in correlation
Microfluidizer m110s, by Microfluidics (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Model 500 ultrasonic dismembrator, by Thermo Fisher Scientific (1 mentions)
Lugol solution, by Merck Group (1 mentions)
Glycogen, by Thermo Fisher Scientific (1 mentions)
Nylon syringe filter, by PerkinElmer (1 mentions)
Centrivap console, by Labconco (1 mentions)
Branson ultrasonic cleaner, by Emerson (1 mentions)
8 protocols using analog vortex mixer
1
Microfluidized Kolliphor EL/Pluronic P105 Emulsions
2
Chlorpyrifos Adsorption Kinetics on Microplastics
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A volume of 50 mL of aqueous chlorpyrifos solution (150 μg L−1) was put in contact with 50.0 mg weight of all the six pristine MP (LDPE, PA6, UPVC, PP, PS, EVA) (corresponding to a concentration of 1 g L−1), in a glass Erlenmeyer flask. The initial samples were collected before adding the MP. Control samples (without MP) were run during the experimental period (96 h). The glass Erlenmeyer flasks were shaken continuously by an orbital (Orbital shaker AO-400, Busen) at 110 rpm, and aliquots of 1.0 mL were collected after 1 min of vigorous shaking in the vortex (VWR—Analog Vortex mixer, Radnor, PA, USA). An aliquot of 1.0 mL of each system was collected and filtered using a PTFE filter (0.22 μm pore size diameter) and a liquid-liquid extraction with n-hexane followed by the CPF determination was performed as described in Section 2.3.3 and Section 2.3.4 , respectively. The removal efficiency was calculated using the same formula as the recovery efficiency (Equation (1)) to compare each MP behavior. The adsorption capacity of the MP was calculated according to Equation (2) for the kinetic studies:
where C0 and Ct are the initial and concentration solution in a determined period (μg L−1), V is the volume of CPF solution at that time t and m (g) is the MP mass.
where C0 and Ct are the initial and concentration solution in a determined period (μg L−1), V is the volume of CPF solution at that time t and m (g) is the MP mass.
3
Vegetable Maceration by Pectinolytic Enzymes
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The ability of microbial pectinolytic enzymes to macerate vegetable was analyzed using the epicarp and mesocarp of cucumber (Cucumis sativus) as solid substrate. Cucumber is a standard model generally used for studying native pulp degradation and maceration capacity of pectinolytic enzymes from microbial culture (Schwan et al., 1997 ; Khan and Latif, 2016 ). Each sample (of cucumber) weighting approximately 5 g was submerged in 10 mL of crude enzyme from the 48 h culture supernatant described above. The submerged sample was incubated for 24 h at 30 °C under shaking at 150 rpm. The negative control consisted of cucumber submerged in PSM (enzyme free). After incubation, the crude enzyme or the PSM was removed, replaced with 10 mL distilled water, and then vortexed at highest speed (VWR, Analog Vortex Mixer) for 1 min. The debris obtained from the macerated cucumber was centrifuged at 100 × g for 5 min.
Pectin degradation was also measured after incorporation of 0.2 % (w/v) apple pectin into 20 % agar, at pH 5.0 (citrate-phosphate buffer) or pH 8.0 (Tris-HCl, buffer). After solidification of the agar, 5 U of enzyme was spotted on pectinized plate agar, incubated 5 h at 30 °C. Then, the solid plate was flooded with lugol solution (Sigma Aldrich, Pennsylvania) to detect clearance zones corresponding to enzymatic activity (Ouattara et al., 2008 ).
Pectin degradation was also measured after incorporation of 0.2 % (w/v) apple pectin into 20 % agar, at pH 5.0 (citrate-phosphate buffer) or pH 8.0 (Tris-HCl, buffer). After solidification of the agar, 5 U of enzyme was spotted on pectinized plate agar, incubated 5 h at 30 °C. Then, the solid plate was flooded with lugol solution (Sigma Aldrich, Pennsylvania) to detect clearance zones corresponding to enzymatic activity (Ouattara et al., 2008 ).
4
Production of Nanoemulsions with Microfluidization
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All nanoemulsions were produced following earlier published protocols18 (link),33 (link),36 (link),38 (link). Briefly, a micelle solution of blended non-ionic surfactants (2% Pluronic P-105, 3% Pluronic P-123 w/v) was made by following previously reported protocol with minor modifications55 (link). To prepare nanoemulsions, micelle solution was added to the mixture of PFC and HC oils, or single (PFC or HC) oil. Then, coarse pre-emulsions were produced with an analog vortex mixer (VWR, Radnor, PA, USA) on high for 30 s. Coarse emulsions were sonicated on ice for 30 s at 29% amplitude (equivalent of 3480 W s) with Model 450 Digital Sonifier (BRANSON Ultrasonics Corporation, Danbury, CT, USA). Microfluidization was performed on all coarse emulsions by Microfluidizer M110S (Microfluidics Corp., Westwood, MA, USA) at 15,000 psi liquid pressure for the specified number of pulses (5 pulses to 1 pass) over an ice-cold interaction chamber. Emulsions were packaged in glass vials and stored at 4 °C.
5
RNA Extraction from Plasma Samples
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A total of 200 µL of plasma were used for each sample, and 800 µL of TRIzol Reagent (ThermoFisher Scientific, cat# 15596018) were added to the sample. The mixture was vortexed for 15 s at high speed (VWR, Analog Vortex Mixer), kept at room temperature for 10 min, vortexed for another 15 s after 200 µL of chloroform were added to it, and kept at room temperature for another 10 min. The mixture was then centrifuged at 12,000× g for 15 min at 4 °C, and the aqueous supernatant was transferred to a new tube. Then, the following reagents were added to the new tube: 700 µL of isopropyl alcohol, 2 µL of glycogen (ThermoFisher Scientific, cat# R0561), and 50 µL of 3M sodium Acetate (PH5.2, Quality Biological, cat# 351035721). The mixture was kept at −80°C until it was frozen (~20 min). Finally, the mixture was centrifuged at 20,000× g for 20 min. The pellet was washed with 1000 µL of 75% pre-cold ethyl alcohol, and was dissolved in 20 µL of nuclease-free water.
6
Quantitative Analysis of Pesticides using LC-MS/MS
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LC System—QSight® LX-50 LC (PerkinElmer, Shelton, CT).
MS system——QSight™ 420 MS/MS detector with ESI and APCI source with HSID interface and Simplicity 3Q™ software platform (PerkinElmer, Shelton, CT).
LC Column—Quasar SP Pesticides C18, 100 mm long, 4.6 mm ID, 2.7 μm particle size, and particle is superficially porous (PerkinElmer, Shelton, CT).
Mixer—analog vortex mixer (VWR, Radnor, PA).
Centrifuge—Eppendorf centrifuge 5430 (Eppendorf Co. Ltd).
Polypropylene centrifuge tubes—15 mL and 50 mL (PerkinElmer, Shelton CT).
Glass volumetric flasks—50 mL (VWR, Radnor, PA).
Syringes—3-mL plastic syringes with Luer lock (Becton Dickinson, Fingerlakes, NJ).
Filter—Nylon syringe filter, diameter 30 mm, 0.22 μm pore size (PerkinElmer, Shelton, CT).
LC vials—2-mL amber glass (PerkinElmer, Shelton, CT).
7
Plasma Protein Precipitation and Extraction
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Plasma samples
were extracted by a standard protein precipitation method (Figure S6A ). A total of 200 μL of plasma
was transferred to a 1.5 mL Eppendorf tube. When appropriate, 50 μL
of the internal standard (from working solution 2000 ng/mL prepared
in 4:1 ACN:MeOH) was added to the sample. Proteins in the matrix were
precipitated by the addition of 550 μL of cold ACN and vortexed
for 10 min using a VWR Analog Vortex Mixer. Samples were then centrifuged
for 10 min at 1000g at 4 °C using a VWR Galaxy
16 microcentrifuge. The supernatant was collected and dried using
a Labconco Centrivap console. Dried samples were reconstituted by
adding 200 μL of 100% MeOH solution, vortexed for 10 min, and
centrifuged at 1000g for 1 min. Samples were transferred
to autosampler vials, sealed, and injected onto the UPC2 system.
were extracted by a standard protein precipitation method (
was transferred to a 1.5 mL Eppendorf tube. When appropriate, 50 μL
of the internal standard (from working solution 2000 ng/mL prepared
in 4:1 ACN:MeOH) was added to the sample. Proteins in the matrix were
precipitated by the addition of 550 μL of cold ACN and vortexed
for 10 min using a VWR Analog Vortex Mixer. Samples were then centrifuged
for 10 min at 1000g at 4 °C using a VWR Galaxy
16 microcentrifuge. The supernatant was collected and dried using
a Labconco Centrivap console. Dried samples were reconstituted by
adding 200 μL of 100% MeOH solution, vortexed for 10 min, and
centrifuged at 1000g for 1 min. Samples were transferred
to autosampler vials, sealed, and injected onto the UPC2 system.
8
EV Degradation Optimization Protocol
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EVs were exposed to the following conditions to determine the amount of degradation. Vortexing: 50 µL of EVs were continuously vortexed for 5 minutes at 2000 rpm using an analog vortex mixer (VWR, Bohemia, NY, USA). Sonication: 50 µL of EVs were placed in a Branson Ultrasonic Cleaner (Model 3510, Branson Ultrasonics Corporation, USA) for 10 min at room temperature. Freeze-thaw cycles: 50 µL of EVs were exposed to 10 freezethaw cycles. This was performed by freezing the samples at -80 • C for 30 seconds and then allowing them to thaw at room temperature for 2.5 min completely. Three technical replicates from each concentration were tested and described in supplementary materials. The optimization of degradation conditions was described in supplementary materials.
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