Ammonium Oxalate

The polysaccharides were extracted as the method from Peng et al. with minor modification [15 (link)]. The crude cell wall material was suspended in 0.5% (w/v) ammonium oxalate and heated for 1 h in a boiling water bath, and the supernatants were combined as total pectin. The remaining pellet was suspended in 4 M KOH containing 1.0 mg mL^-1 sodium borohydride for 1 h at 25°C, and the combined supernatant was neutralized, dialyzed and lyophilized as hemicelluloses. The KOH non-extractable residue was further extracted with acetic-nitric acids for 1 h at 100°C and the remaining pellet was defined as crystalline cellulose. All samples were carried out in triplicate for wall fractionations.

Plaque assays to compare the transgenic parasites with their parental strains were performed on HFFs cells in 6-well tissue culture plates (Corning costar, Beijing). Briefly, 500 parasites per well were seeded into confluent monolayers and infected cells were maintained in fresh DMEM containing 10% FBS and incubated undisturbed at 37°C in 5% CO₂ for 7 days. To stain the monolayers, media was aspirated and disassociated parasites were washed off using PBS. Cell monolayers were then fixed for 10 minutes in PBS with 4% formaldehyde and stained with crystal violet solution (12.5 g crystal violet dissolved in 125 mL ethanol and mixed with 500 mL 1% ammonium oxalate in water) at room temperature for 10 minutes, washed with deionized water, air dried and visualized by microscopy using image acquisition and plaque area measurement as previously described [42] (link).
To analyze the intracellular growth rate of the transgenic parasites compared with their parental strains, about 1×10⁵ parasites were inoculated on confluent HFFs in 24-well plates. The infected cells continuously incubated for 24 hours. Thereafter, cells were fixed in PBS with 4% formaldehyde, and RH and RHΔKU80 parasites were stained for IFA using rabbit anti-SAG1 polyclonal antibody as described above. TgHMGB1a overexpression and B box-deficient parasites were directly examined for fluorescence (GFP and eGFP, respectively). The numbers of parasites per vacuole for a minimum of 150 randomly chosen vacuoles were counted for each strain using a fluorescence microscope (IX71, Olympus, Japan) at 400× magnification.

The photocatalytic behaviour was investigated by the oxidation of methylene blue and reduction of Cr(VI). The photocatalytic performance was evaluated under simulated solar light using a 300 W Xe lamp (a high-pressure 150 W xenon lamp, LOT – QuantumDesign GmbH equipped with the AM1.5G filter). The intensity of the incident light that reaches the surface of the investigated solution was equal to 100 mWcm⁻² (measured using a Coherentâ FieldMate Laser Power Meter). In a typical test, 20 mg of catalyst was placed in a 50 mL aqueous pollutant solution. The concentration of MB and Cr(VI) was 1·10^–5 M. Before irradiation, the suspension was vigorously stirred in the dark for 30 min to reach desorption-adsorption equilibrium. The change in MB and Cr(VI) concentration was monitored by its absorption at 665 nm and 351 nm, respectively, from the UV–Vis (Spektrofotometr UV5100) spectra of the solution, using distilled water as a reference. A total of 0.75 ml of suspension was collected and centrifuged before UV‒Vis measurement. In the case of Cr(VI) photoreduction, the process was conducted in acidified (pH = 3) solutions.
To study the reusability of the prepared photocatalysts, the cycle experiment was repeated 4 times for the photodegradation of methylene blue. After each photodegradation test, the catalyst was collected by centrifugation, dried under natural conditions and used for the next degradation experiment. Moreover, to indicate the role of hydroxyl radicals (·OH), (h +) holes and superoxide radicals (·O2-) in the process of MB degradation, experiments were performed in the presence of appropriate scavengers: t-butanol (TBA), ammonium oxalate (AO) and benzoquinone (BQ). The concentration of each scavenger was equal to 1 mM.

Ammonium metavanadate (NH₄VO_3, 99.0%), oxalic acid dihydrate (C₂H₂O₄ × 2H₂O, 97.0%), and methylene blue (MB > 98%) were obtained from Sigma‒Aldrich and used without further purification. Deionized water was used in all experiments (conductivity < 0,06 μS/cm). Graphene oxide (GO) employed in the composite synthesis was prepared using the modified Hummers method ^{69 (link)}. Potassium dichromate (K₂Cr₂O₇, ≥ 99.0%) and ammonium oxalate (AO, ≥ 99%) were purchased from Merck. Benzoquinone (BQ, > 98%) and tert-butyl alcohol (TBA, > 99.5%) were received from CheMondis.

The production of H₂O₂ by BSA-GOx-NPs, glucose, and oxygen was determined via a classic colorimetric method, applying ammonium titanyl oxalate as the indicator. In brief, 0.1 mL of BSA-GOx-NPs or BSA-NPs suspension (50 μg/mL BSA) was mixed with 0.1 mL of different glucose concentrations (0, 0.025, 0.05, 0.1, 0.2, 0.5, 1 and 2 mg/mL). After 1 h, ammonium titanyl oxalate solution (10 μL, 10 mM) was added. The obtained yellow suspension was measured by a microplate reader (Nano Quant, Tecan) at 405 nm.