Ptfe filter

100 μL of each sample was diluted 1 : 10 in 50% methanol : water (Honeywell, Chromasolv™) and analysed spectrophotometrically at 280 nm in a 96-well UVStar® plate (Greiner Bio-One) and compared to a standard curve of Sigma Kraft Lignin (Sigma Aldrich, 471003), Fig. S1,† to determine the overall aromatic content. Concentrated samples were filtered using a 0.2 μm PTFE filter (VWR) and diluted in 50% methanol : water to obtain a final concentration of 150–175 μg mL⁻¹ lignin.

Prior to analysis, all samples were filtered through a PTFE filter (VWR, Leuven, Belgium). Naringenin and p-coumaric acid were quantified using a Waters Acquity UPLC H-Class system connected to an ACQUITY TUV-detector operating at 30 °C and 290 nm. A Kinetex^® 2.6 µm Polar C18 100 Å column (Phenomenex, Utrecht, The Netherlands) was used to separate metabolites using the following method, at a flow rate of 0.6 mL/min:

Time (min)	Eluent A: 0.1% TFA in water (%)	Eluent B: 100% acetonitrile (%)
0	90	10
0.5	75	25
5	75	25
7	30	70
8.5	30	70
10	90	10

Glycerol was quantified on a Shimadzu Prominence-I LC2030c Plus system connected to an RID-20A (Shimadzu) detector operating at 40 °C. A Rezex ROA-Organic Acid H + (8%) – 150 × 7,8 mm column (Phenomenex, Utrecht, The Netherlands) at 60 °C was used to separate metabolites using an isocratic method with a flow rate of 0.6 mL/min and 0.005 N H₂SO₄ in water as eluens.

Samples were weighed in a 15 ml polypropylene tube (Falcon tubes, VWR International) to achieve a 1/10th dilution and then kept on ice. Cereulide was extracted by using acetonitrile (LC-MS grade, Merck) as described in the ISO 18465 (2017) . An internal marked standard (¹³C₆-cereulide) was added to reach a final concentration of 1.5 ng/ml.
The tube was then vigorously horizontally shaken at room temperature on an Orbital shaker (VXR basic Vibrax, VWR International) for circa 1 h at around 1,800 rpm (until adequate vortexing was achieved). After shaking, the tube was centrifuged (centrifuge Sigma-Aldrich) for 10 min at 1,000 G. The supernatant was filtered using a 0.22 μm Polytetrafluoroethylene (PTFE) syringe filter (PTFE filter, 0.22 μm, 13 mm minispike, VWR International) into a 2 ml amber glass vial (Agilent). The filtered extract was stored at -20°C until the injection.

Supernatants obtained from centrifuged cultures were filtered through 0.2 μm polytetrafluoroethylene (PTFE) filters (VWR, Barcelona, Spain). BA were derivatized with diethyl ethoxymethylenemalonate (DEEMM) (Sigma–Aldrich). 100 μl of sample were mixed with 175 μl of 1 M borate buffer (1 M boric acid neutralized with NaOH until pH 9.0), 75 μl of methanol (Merck), 2 μl of L-2-aminoadipic acid as internal standard (2 g/L) (Sigma–Aldrich) and 3 μl of DEEMM. The mixture was incubated at 30°C in an ultrasound bath (Selecta, Barcelona, Spain) for 45 min. Samples were then heated at 70°C for 2 h to allow the complete degradation of excess DEEMM and reagent by-products. Samples were filtered through 0.2 μm PTFE membranes (VWR) before injection into the chromatograph system. Samples were diluted, when necessary, with 0.1 N HCl (Merck). BA were separated and quantified by ultra high performance liquid chromatography (UHPLC) system (Waters, Milford, MA, United States) with an UPLC^®BEH C18 1.7 μm column (Waters), following the method previously described (Redruello et al., 2013 (link)). Empower 2 software (Waters) was used to control the system and to analyze the data. Standards were prepared with agmatine, putrescine dihydrochloride (Acros Organics, Geel, Belgium), tyrosine and tyramine in Milli-Q water. The BA concentrations provided are the average of three independent cultures.

To determine the binding affinities of the MIP/NIP, rebinding experiments were conducted as follows: to 20 mg of MIP/NIP powder was added 5 mL aliquots of aqueous target/analogue ranging in concentration (0.1–0.7 mM) and the resulting suspension agitated on a rocking table (125 rpm) for 90 min. After agitation, the filtrate (0.45 µm pour size, PTFE filters, VWR, cat number: 514-0065) of each sample was collected and analyzed using a UV-spectrophotometer, analyzing the λ_max of the remaining molecular species in solution (C_f). The amount of molecular species bound to the MIP/NIP (S_b) proceeded to be calculated from these observed values and the corresponding binding isotherm plotted. This process was repeated for each of the following species: Amoxicillin, ampicillin, cloxacillin, malachite green, crystal violet, and mordant orange 1.
Note: During the experimentation with malachite green, all samples of crystal violet and mordant orange were diluted (dilution factor 100), ensuring absorbance values within the reliable range on the UV-spectrophotometer.

A dose-response study was constructed by incubating the synthesized dye loaded MIP 205 (40 mg) with 5 mL of aqueous amoxicillin at varying concentrations (0.000–365.0 mg L⁻¹) for 1 min before the filtration (0.45 µm pour size, PTFE filters, VWR, cat number: 514-0065) of the sample. The filtrate proceeded to be analyzed by a UV-spectrophotometer collecting the spectrum for each sample (200–500 nm). The maximum absorbance of each concentration (λ_max = 385 nm) was then plotted against the concentration of amoxicillin added, allowing the dose response to graphed. The same process was repeated for the assay in the presence of cloxacillin and ampicillin, testing the selectivity of the assay.