Waters 2695

To analyse the changes in physiological indexes of S. miltiorrhiza plants under drought stress, total protein, superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA), proline (PRO), and catalase (CAT) were determined using commercial kits purchased from the Jiancheng Bioengineering Institute (Nanjing, China) [33 (link)–35 (link)]. Table S 1 in the supplementary material displays all of the measurement method of these physiological indexes.
The main tanshinone contents in the roots of S. miltiorrhiza plants treated with different drought stress were determined using high performance liquid chromatography (HPLC, Waters 2695, USA). It was performed according to the previous method in our laboratory. All of the roots of samples were freeze-dried for 48 h, and then grind to a powder using a mortar. The 0.25g sample was extracted in 25 mL 100% methyl alcohol and subjected to ultrasonic shock for 50 min. The extracts were centrifuged at 4000 rpm for 10 min and later filtered through a 0.22 μm microporous membrane (Jinteng, Tianjin China) for analysis.

The 3,5-dinitrosalicylic acid (DNS) method was used to determine the reducing sugar content. The ethanol content was assayed using gas chromatography (GC) as described by Liang et al. [19 (link)] with slight modifications. The sample was analyzed on a GC system (Agilent Technologies, Inc., Palo Alto, CA, USA), and a DB-Wax column (30 m × 0.25 mm, 0.25 μm film thickness, Agilent Technologies, Inc., Palo Alto, CA, USA) was used, with column and injector temperatures of 60 °C and 200 °C, respectively.
The GABA content was quantified using high-performance liquid chromatography (HPLC) (Waters 2695, Waters corp., Reno, NA, USA) as previously described [14 (link)]. The separation column was an Inertsil ODS−3 C18 column (4.6 mm × 150 mm, 5 μm, Shimadzu, Japan). The mobile phases used were A (0.05 M sodium acetate: methanol: tetrahydrofuran, 84: 15: 1, V/V/V) and B (methanol). The flow rate and temperature of the elution phase were maintained at 1.0 mL/min at 30 °C.
The organic acid analyses were also performed using HPLC as previously described [14 (link)]. The separation column was an ionic exchange resin Bio-Rad Aminex HPX−87H column (7.8 mm × 300 mm, 9 μm; Bio-Rad Laboratories, Inc., Hercules, CA, USA) with a flow rate of 0.6 mL/min and 5 mmol/L sulfuric acid as the mobile phase. The UV detection wavelength was 210 nm, and the column oven temperature was maintained at 60 °C.

Free amino acids in young shoots samples (100 mg) were extracted by 5 mL boiling water for 5 min in 100°C water bath. Amino acid contents were measured using an automatic amino acid analyzer (Sykam S-433D, Germany) [23] . Standards were prepared from authentic reagents (Sigma-Aldrich Co., St. Louis, MO).
For measurement of chlorophylls and carotenoids, plant samples were extracted with acetone at 4°C for 16 h in the dark. Their concentrations were determined by means of HPLC (Waters 2695, Waters Corp. USA). A volume of 20 µL extract was injected into Phenomenex synergi Hydro-RP C₁₈ column (250 mm×4.6 mm, 4 µm) kept at 35°C and eluted with solutions A and B with gradients running at 1 mL/min as previously reported [24] . Solution A was a mixture of acetonitrile, acetic acid, water at a volume ratio of 3∶0.5∶96.5 and B was a mixture of acetonitrile, methanol and chloroform at a volume ratio of 75∶15∶10. Solution B increased from 80% to 100% in the first 20 minutes and was then held at 100% for the next 15 minutes. Absorbance was recorded at 450 nm by a photodiode array detector (Waters 2998, Waters Corp. USA) and pigments was identified by comparing retention time and absorption spectra or authentic reagents (lutein and β-carotene, Sigma-Aldrich Co., St. Louis, MO) [25] .

Liquid samples to determine sugar, inhibitor and extracellular metabolite concentrations in the MBR were taken from the permeate outflow from the cross flow membrane unit and stored at −20 °C until analysis. Quantification of the compounds was performed by HPLC (Waters 2695, Waters Corporation, Milford, MA, USA) using an Aminex HPX87-H column (Bio-Rad Laboratories, München, Germany) for extracellular metabolite and inhibitor concentrations, and an Aminex HPX87-P column (Bio-Rad Laboratories, München, Germany) for sugar concentrations. A refractive index detector (Waters 2410, Waters Corporation, Milford, MA, USA) was used to detect all components. The Aminex HPX87-H column was maintained at 60 °C and eluted with 5 mM H₂SO₄ at a flow rate of 0.6 mL·min⁻¹, whereas the Aminex HPX87-P column was kept at 85 °C using MilliQ water at an eluent flow rate of 0.6 mL·min⁻¹.

To understand the fermentation processes, seven fermentation parameters, including temperature, moisture, acidity, reducing sugar, alcohol, acetic acid, and lactic acid were detected. The temperatures of the sampling locations were measured and recorded by electron probe thermometer before sample collection. Moisture of fermented grains was determined by a gravimetric method by drying samples to a constant weight at 125°C. The acidity was measured based on the methods described by others (Tan et al., 2019 (link)). Alcohol, reducing sugar, and organic acids were analyzed via high-performance liquid chromatography (HPLC; Waters 2,695, Milford, United States) equipped with refractive index detector (RID, 2414) and photodiode array detector (PDA, 2998), based on the method described elsewhere (Zheng et al., 2014b (link); Wang et al., 2017 (link); Jiang et al., 2019 (link)).

The amino acid sequence of affibody molecule Z_hcHER2:342 was MIHHHHHHLQVDNFNKEMRNAYWEIALLPNLNNQQKRAIRSLYDDSQSANLLAEAKKLNDAQAPKVDC.
The cF₁₃-affibody conjugate was constructed according to our previous report [38 (link),39 (link)], and the synthetic route is described in Scheme A2. The obtained DNA-affibody conjugate was analyzed and characterized by UV-vis spectroscopy and 2% agarose gel electrophoresis. The molecular weights of cF₁₃-affibody and affibody were also detected by gel permeation chromatography (GPC, Waters 2695, Milford) using polystyrene as standard. The pure cF₁₃-affibody was concentrated using Amicon ultracentrifugal filters (MWCO 10 kDa, Merck Millipore, Darmstadt, Germany) and stored at 4 °C.