2.5. Fluorescence Measurements

Concentrations of H₂O₂ to be measured were in the range of 0.1–10 mM. Data were collected from the fluorescence intensity of the H₂O₂-sensing membrane at two emission wavelengths (λ_em = 500 nm and λ_em = 560 nm) with an excitation wavelength of 400 nm (λ_ex = 400 nm). Fluorescence spectra for the detection of H₂O₂ were measured with a multifunctional fluorescence microplate reader (Safire², Tecan Austria GmbH, Wien, Austria). The immobilization efficiency of HRP in the HRP–QD–AF membrane was calculated by dividing the amount of immobilized HRP by the total amount of HRP used for immobilization. The amount of the immobilized HRP was determined by subtracting the amount of unimmobilized HRP from the total amount of HRP used. Unimmobilized HRP was separated from immobilized HRP in one well by washing several times with 1.0 mL of 10 mM phosphate buffer (pH 7.4). Protein concentrations of the washed, unimmobilized HRP were determined with the Bradford method. Optimization of HRP amount for immobilization was performed with 1, 5, 10, 15, and 20 units (U). Sensitivities of the HRP–QD–AF membranes immobilized with different amounts of HRP were evaluated based on the slope value (sensitivity index (SI)), i.e., the ratio of fluorescence intensities at two emission wavelengths (λ_em = 500 nm and λ_em = 560 nm) considering H₂O₂ concentration. Kinetic parameters (K_m and V_max) of the immobilized HRP were determined from the Lineweaver–Burk plot based on the ratio of fluorescence intensities at λ_em = 500 and λ_em = 560 nm.

Reversibility of the H₂O₂-sensing membrane was performed at 0.1, 1.0, and 10 mM H₂O₂. The H₂O₂-sensing membrane was measured in a sequence of H₂O₂ concentrations (from low to high concentrations), and the measurement cycle was repeated. A multifunctional fluorescence microplate reader was set for fluorescence measurements against time with an interval of 30 s during 10 min.

Effects of pH and temperature on H₂O₂ measurements were investigated. Solutions of 1 mM H₂O₂ with pH in the range of pH 5.0 to pH 9.0 were exposed to the H₂O₂-sensing membrane. The H₂O₂-sensing membrane was also tested with different temperatures (25, 30, 33, 35, 37, and 40 °C) for H₂O₂ concentrations ranging from 0.1 to 10 mM. The long-term stability of the H₂O₂-sensing membrane was evaluated by determining its repeatability by measuring the fluorescence intensity at various H₂O₂ concentrations after a number of measurements.

Response of the QD–AF membrane to H₂O₂ was used to detect α-ketobutyrate according to the following reaction:

Residual amounts of H₂O₂ were expressed via fluorescence quenching of the QD–AF membrane after a fixed amount of H₂O₂ (10 mM) reacted with 1 mL of different concentrations of α-ketobutyrate. These quenched fluorescence intensities corresponded to concentrations of α-ketobutyrate. Concentrations of α-ketobutyrate were also colorimetrically determined by fluorescence quenching of the QD–AF membrane. The QD–AF membrane was exposed to solutions resulting from the reaction of H₂O₂ with different concentrations of α-ketobutyrate. Changes in the color of the QD–AF membrane were obtained using a microscopic fluorescence camera (AM4115T-GRFBY Dino-Lite Edge: λ_ex = 470 nm and λ_em = 510 nm, AnMo Electronics Co., Taipei, Taiwan) placed in a homemade black chamber.

Concentrations of H₂O₂ in artificial wastewater were also determined using the HRP–QD–AF membrane. The artificial wastewater solution containing 2.5 mM CaCl₂, 45 mM NaCl, 3.5 mM KH₂PO₄, 3.5 mM K₂HPO₄, 2.5 mM NaHCO₃, 1 mM MgSO₄, 2.5 mM Na₂SO₄, and H₂O₂ at different concentrations (0.1–10 mM) was prepared.