Spectrofluorometer

Plasma and urinary LPO levels were measured as thiobarbituric acid-reactive substances^{20 (link)}. Briefly, plasma or urine aliquots were mixed with 8% SDS and a mixture of 0.8% 2-thiobarbituric acid and 20% acetic acid. These mixtures were incubated at 95 °C for 60 min. After cooling, the samples were centrifuged at 1600 × g for 5 min to precipitate interfering particulate materials. The LPO level was measured using a spectrofluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The LPO concentration in the kidney was measured using an LPO assay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions.

The Annexin V‐FITC/PI Apoptosis Detection Kit (Solarbio, Beijing, China) was used to detect apoptotic cells. Cells were collected 24 hours after treatment, washed with PBS (Solarbio, Beijing, China) and resuspended in binding buffer (500 μL) in an Eppendorf (EP) tube. Annexin V‐FITC and PI (5 μL) were added into binding buffer. After mixing, the EP tube was kept away from light for 5‐15 minutes at room temperature. Flow cytometry (BD, San Diego, CA, USA) was used to identify cells of normal status, early apoptosis, late apoptosis and death; FITC was detected using channel FL1, and PI was detected using channel FL3.
Caspase‐3/7 activity was measured using the Apo‐ONE^® Homogeneous Caspase‐3/7 Assay kit (Promega, Madison, WI, USA). Cells were seeded in 96‐well plates and cultured with baicalein (0, 40, 80 μmol/L) for 24 hours. Apo‐ONE^® Caspase‐3/7 reagent was added (100 μL) and mixed with medium, and the plate covered with a plate sealer and incubated for 3 hours. The fluorescence (RFU) of each well was measured using a spectrofluorometer (Thermo Scientific) at an excitation wavelength of 499 nm and an emission wavelength of 521 nm. Caspase‐3/7 activity = RFU_{(treatment group)}/RFU_{(control group)} × 100%.

Cyclic voltammetry (CV) and different pulse voltammetry (DPV) were performed using a Dropsense potentiostat (Methrohm, Singapore). The 17β-Estradiol (E2) aptasensor was fabricated on the screen-printed electrode (SPE) (Scrint Technology (M) Sdn. Bhd) and was used as working electrode. A rod-shaped glassy carbon electrode and Ag/AgCl electrodes (containing 3.0 M KCl) were used as auxiliary and reference electrodes with scan rate of 0.05 mV/s, respectively. The potential used ranged from −1.0 V to 1.0 V was applied in all measurements. All homogeneous mixture of material solutions were prepared using sonicator bath Elma S30H. The morphology and chemical characterizations of nanowires and microspheres were measured using Scanning Electron Microscope (SEM) (ZEISS, Jena, Germany) and Fourier Transform Infrared (FTIR) (Perkin Elmer, Massachusetts, United State). The fluorescence spectra were measured using spectrofluorometer (Thermo Fisher Scientific Inc., Wilmington, NC, USA).