F 4600 spectrofluorophotometer

Bovine serum albumin (BSA) and chloroauric acid (HAuCl₄.3H₂O) were purchased from Sigma-Aldrich and Shanghai Reagent (Shanghai, China), respectively. Glutaraldehyde, bilirubin, and all other reagents used in the experiment were obtained from Aladdin Reagent (Shanghai, China). Bilirubin (1 mg) was first dissolved with 0.1 M NaOH (0.1 mL) and then diluted with 50 mM phosphate-buffered solution (PBS) (pH 7.4) to 10 mL to obtain a stock solution (171 μM). Chemicals and solvents were all of analytical reagent grade unless otherwise stated. Ultrapure water (18.2 MΩ, Milli-Q, Millipore) was used throughout the experiment.
Fluorescence spectra and UV-vis absorption spectra were measured with a Hitachi F-4600 spectrofluorophotometer and a Hitachi UH-5300 spectrophotometer, respectively. Scanning electron microscopy (SEM) images were obtained from a field emission SEM (Hitachi S-4800). The morphology and size of the gold nanoclusters were characterized by a JEOL 2100 high-resolution transmission electron microscope (HRTEM) operating at an accelerating voltage of 200 kV.

A previously construct of N39-CFP-C-YFP (designated as D2-CFP-YFP in this study) was also used as a template for generating mutant N463r-CFP-YFP (Meng et al., 2017 (link)), in which TM XIII was replaced with the corresponding region of NhaD1, CFP was fused after site 39 and YFP was fused at the C-terminal of N463r. In these two fusion proteins, CFP and YFP were fused after site 39 and C-terminus, respectively. E. coli KNabc carrying these plasmids were tested for antiport activities. After activity confirmation, E. coli C43 strains carrying these fusion plasmids were grown at 37°C in LB medium to a concentration (OD₆₀₀ = 0.6–0.8) and induced by adding IPTG (final concentration of 0.5 mM), and cultivated at 20°C overnight. Cells were harvested and washed three times with the Tricine-KOH buffer solution (10 mM Tricine and 140 mM KCl, adjusted to pH 8.5 with KOH), and resuspended in the same buffer to an OD₆₀₀ of 1.5. For fluorescence resonance energy transfer (FRET) analysis, a 2-ml cell suspensions of D2-CFP-YFP, N463r-CFP-YFP, or D2-CFP were irradiated at 433 nm to excite CFP, and recorded the fluorescence emission at 450–600 nm on a F-4600 spectrofluorophotometer (Hitachi). In the meanwhile, a 2-ml cell suspension of D2-CFP-YFP was also illuminated at 473 nm and recoded at 450–600 nm.