Nile red

The copolymers of PLA_5K-PAE_5K-mPEG_5K, PAE_5K-PLA_5K-mPEG_5K and PLA_5K-PEG_5K-PAE_5K were purchased from Ruixi Biological Technology Co., Ltd (Xi’an, Shanxi, China). The details of synthesis and characterization of the copolymers were shown in the Additional file 1. Biotin LPS and A LIVE/DEAD^®BacLight™ Bacterial Viability Kit were obtained from Nanocs, Inc. (New York, USA) and Thermo Fisher Scientific Inc. (Shanghai, China), respectively. Nile red and Triclosan (TCS) were obtained from Macklin Biochemical Co., Ltd (Shanghai, China).

Goose liver was purchased from Goose Co. Ltd., Ningbo, China. KGM, SPI, sodium hydroxide (NaOH), Fluorescein isothiocyanate (FITC), Nile red, dimethyl sulfoxide (DMSO) alkaline protease, ether petroleum ether, chloroform, glacial acetic acid, potassium iodide (KI), potassium bromide (KBr), soluble starch, sodium thiosulfate (Na₂S₂O₃), hydrochloric acid (HCl), trichloroacetic acid (C₂HCl₃O₂), thiobarbituric acid (C₄H₄N₂O₂S), pepsase, trypsin and n-hexane were reagent grade and purchased from MACKLIN Co. Ltd., Shanghai, China.

The samples from the anammox reactor were pretreated by staining and then observed by CLSM to detect the EPS (proteins, β-D-mannose, and lipid). The procedures of staining were adopted from a reference (Wu et al., 2018 (link)) and with a little modification that is described as follows: (1) Fluorescein isothiocyanate (FITC) (CAS No. 27072-45-3, Macklin, Shanghai, China) was applied to stain proteins; (2) Calcofluor white (CalW) (CAS No. 4404-43-47, Sigma-Aldrich Corporation, California, CA, United States) for β-D-mannose detection; and (3) Nile Red (CAS No. 7385-67-3, Macklin, Shanghai, China) was used to stain lipids.
The detailed staining steps were followed by adding a drop of FITC (10 mg/L, dimethyl sulfoxide as solvent) to the samples standing for 30 min and washing for three times by using phosphate-buffered saline (PBS) (0.01 M, pH = 7.4). Then, using CalW solution combined with 10% KOH (1:1) stained 1 min and washed with PBS several times. Next, put the samples in Nile Red solution (5 mg/L, methanol as solvent) to stain for 5 min. Finally, a drop of the antifade mounting medium was added to the test samples. After staining, using PBS washing then put on the glass slide and observed by CLSM (Leica TCS SP8, Germany) and amplified about 4–10 times. The measuring parameter and observable color from different staining conditions are given in Table 2.

Quinoa seeds and soybean oil were purchased from Haishang International food franchise store and Yihai Kerry Arawana Holdings Co., Ltd. (Beijing, China). Nile Blue A, Nile Red and 1, 2-propylene glycol were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). ProClin™150 formaldehyde was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Total protein (TP) assay kit (A045-2-2) was purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). All other chemicals were of analytical grade and purchased from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China).

Zein (from Zea mays L.Cat No.: 294946) was purchased from J&K. Sodium alginate (AR, 90%, S817374) was purchased from Macklin. Aminase bioassay test Kit (MAK310) was obtained from Sigma Aldrich Inc. (St. Louis, MO, USA). Soybean oil was purchased from the local market. Nile Red and Nile Blue A were purchased from Macklin Biochemical Co., Ltd. (Macklin, Shanghai, China). All other chemicals were of analytical grade.

Poly(ethylene glycol) (PEG, Mw ~8000), Poly(vinyl alcohol) (PVA, Mw: 89,000–98,000, 99% hydrolyzed), Poly(2-ethyl-2-oxazoline) (PEtOx, Mw~50, 000), sodium citrate, Poly(ethylene glycol) diacrylate (PEGDA, Mn~575), sodium alginate, 2-Hydroxy-4′-(2-hydroxyethoxy)−2-methylpropiophenone (Irgacure 2959), N,N′-methylenebisacrylamide (MBAA), were purchased from Sigma-Aldrich. Dextran (DEX, Mw ~10,000, 40,000, and 500,000) and Nile red were purchased from Macklin. Acrylamide and lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP) were purchased from TCI. Rhodamine 6 G, Fluorescein isothiocyanate (FITC) labeled Poly-l-lysine (FITC-PLL, Mw ~30,000–70,000), and Fluorescein isothiocyanate–dextran (FITC-dextran, Mw ~40, 000) were purchased from Sigma-Aldrich. Rhodamine-labeled PEG (Rh-PEG, Mw ~40,000) was purchased from Aladdin. Nucleic acid sequences of Cy5-DNA were synthesized by Integrated DNA Technology (IDT). Calcein was purchased from J&K scientific. Millipore Milli-Q water (18.2 MΩ, pH = 7) was used in all experiments. All materials were used as received without further purification.