Sorvall lynx 4000

All samples were collected as a midstream portion of the second morning urine (~60 mL); samples were transferred on ice to the lab and processed according to Thomas et al. [5 (link)] with slight modifications. Briefly, 45 mL of each urine sample was transferred to a 50 mL conical tube and centrifuged at 1500× g for 12 min (Sorvall Lynx 4000, Thermo Scientific, San Jose, CA, USA) to pellet cells. Fifteen milliliters of supernatant was then spun at 10,000× g for 12 min and supernatants were transferred to new tubes. Ten milliliters of urine sample of five individuals per group were pooled in a 50 mL conical tube, aliquoted and stored at −40 °C until analysis.
Human urine sample from one healthy donor was selected for FCSNP test (proof of concept) using single and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE). Additionally, pooled urine from each group of individuals (as indicated before) were selected for MS analysis.

The sample was homogenized using a grinder (T 25 digital ULTRA-TURRAX^®, IKA, Staufen, Germany). After weighing 10 g of the sample, 10 mL of acetonitrile was added to each weighed sample and shaken for 1 min. Thereafter, a QuEChERS extraction kit (magnesium sulfate: 98.5–101.5%; sodium chloride: ≥99.5%; sodium citrate: 99.9%; disodium citrate sesquihydrate: 99%) was added to the sample solution, followed by vigorous shaking for 1 min using a rotary mixer (DE/VIVA, Collomix GmbH, Gaimersheim, Germany). Subsequently, centrifugation was performed for 10 min at 4000 rpm using SORVALL LYNX 4000 (Thermo Scientific, Waltham, MA, USA). Then, 1 mL of the supernatant was put into the QuEChERS dispersive SPE kit (primary secondary amine, octadecyl silane end-capped, magnesium sulfate; 98.5–101.5%), mixed with Mixmate 5353 (Effendorf, Hamburg, Germany) for 1 min, and centrifuged again with Minispin plus 545 (Effendorf, Hamburg, Germany) at 10,000 rpm for 1 min. The liquid separated through this process was filtered with a 0.2 μm PTFE syringe filter (Whatman, Maidstone, UK) and was used as the final sample.

ChNC were prepared by acid hydrolysis of crude chitin powder in 3 M hydrochloric acid (HCl) at 80 °C for 90 min under magnetic stirring. The hot mixture was cooled on ice to stop the reaction. The HCl was removed by centrifuging the samples at 4500× g for 5 min (Sorvall Lynx 4000, Thermo Fisher Scientific, Waltham, MA, USA), after which the supernatant was discarded, and the pellet was redispersed in an equal amount of MilliQ. This step was repeated three times. Subsequently, a 30 mL sample was added to a 50 mL reaction tube, and a sonification step was applied (5000 J energy input with an SFX150, Branson Ultrasonics, Brookfield, CT, USA) to increase chitin nanocrystal yield. Samples were diluted 10 times and centrifuged at 1200× g for 20 min. The supernatant containing the chitin nanocrystals was collected, and the pellet containing the amorphous chitin was discarded. A final centrifugation step was applied at 4500× g for 5 min to concentrate the chitin nanocrystal solution. The chitin nanocrystals were freeze dried before further usage (Christ Epsilon 2-6D, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). For this, samples were frozen at −40 °C for 12 h. The freeze-drying process was performed under a 1030 mbar vacuum. The temperature was increased from −20 °C to +20 °C in 8 stages during the freeze-drying process, which lasted for a total of 48 h.