Micropipette

The pupicidal activity of EOCs was determined by adopting the modified protocol by Zhang et al. [31 (link)], using the topical application method, and the pupae were separated by soft camel hair bush into groups of similar size and healthy. Two days-old pupae (n = 20) randomly selected from the cohort were then treated with 10 µL of each concentration of specific EOCs using an Eppendorf^®micropipette (10−100 μL), and for control treatment, only acetone was applied topically. The treated and control pupae were put in the same 12 cm disposable Petri dishes with a layer of half-filled soil moisture of 70–80%. The pupae were placed in the same control conditions. After 7–10 days of EOC exposure, un-emerged or deformed treated pupae were recorded. Each treatment was replicated three times.

Deltamethrin (Decis^®; 25 g L⁻¹; Bayer Vapi Private Limited-Plot, São Paulo, Brazil) was diluted in 50 mL of distilled water for six concentrations (0.39, 0.78, 1.56, 3.12, 6.25, and 12.5 mg mL⁻¹), where the maximum dilution was equivalent to 125 g a.i./L, corresponding with the field dose recommended for S. frugiperda. Water (distilled and deionized) was used in the control. Concentrations were prepared to evaluate the toxicity (acute or chronic) and determine the relevant toxicological endpoints, following the concentration–mortality relationship and lethal concentrations (LC₂₅, LC₅₀, LC₇₅, and LC₉₀) of this insecticide. Caterpillars were starved for 2 h before starting bioassay. Each solution (1 μL) was applied into 125 mg of artificial diet using an Eppendorf micropipette (1–10 μL, Eppendorf, Hamburg, Germany), which was supplied for the caterpillars during the first day. After this, for caterpillars feeding on the untreated or treated diet by 24 h, a new artificial diet without insecticide was supplied during the evaluation time of the experiment. Thirty third-instar caterpillars of S. frugiperda were individualized in Petri dishes (90 × 15 mm) and used for each concentration bioassay with three replications, following a completely random design. After five days of exposure, caterpillars were counted as dead if they were unable to walk when prodded with a brush.

To prepare the modified GPH-MnPc/SPE biosensor, several stages were covered. Initially, 10 μL of 10⁻⁵ M manganese phthalocyanine solution in chloroform was added—sequentially, with pauses for drying, through the drop-and-dry technique—to the surface of the GPH/SPE. Drying was performed at room temperature for 30 min. An Eppendorf micropipette was used to add the manganese phthalocyanine solution.

The UPLC system consisted of a Photo-Diode Array (PDA) detector, column compartment, autosampler manager, and a binary solvent delivery pump, connected to Waters Empower two software. Micropipette (Eppendorf, Germany), Ultrapure distilled water was prepared using a Millipore Milli-Q-Plus system (Millipore, Bedford, MA, United States). Screening kits for COX-2 inhibitors were purchased from Beyotiome Biotechnology (Shanghai, China). The fluorescence values were measured by Fluorescence microplate reader (GEMINIXS, United States) and the SOFTmaxPRO software was the production of Molecular Devices Company in United States, Ultrasound (Nanjing, China).

The full-wave multifunctional enzyme-labelling instrument (Tecan (Shanghai) Trading Co., Ltd.); micropipette (Eppendorf AG); ultrapure water analyser (model: millibo direct Q-5); vortex instrument (Shanghai Luxi brand instrument company); DMSO (Batch no.1121e037, Beijing Solebao Technology Co., Ltd.); freeze dryer (model: Christ alpha) 1-2 ldplus); 1/100000 scale (Sedolis Scientific Instruments Co., Ltd.); 96-well blackboards (Batch No. 15419010, Corning Co., Ltd.); centrifuge tube; and COX-2 Inhibitor Screening Kit were purchased from Beyotime Biotechnology (Shanghai, China); the Shiyifang Vinum was provided by the First Affiliated Hospital of Guangxi University of traditional Chinese medicine; the quercetin and kaempferol were purchased from the National Institutes for Food and Drug Control (Beijing, China); the luteolin and apigenin were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Chengdu, China).

The modification procedure is consistent with the literature and similar to that used in another previous work [83 (link),84 (link)], using carbon-based screen-printed electrodes (SPE-C) as the substrate. The diameter of the working electrode was 0.4 cm, resulting in a geometric area of 0.1257 cm².
The single-layer carbon nanotubes (SWCNT) suspension was prepared by adding 10 mg single-layer carbon nanotube powder to 10 mL of solvent (a mixture of dimethylformamide: water (1:1)) followed by sonication for 60 min at 59 kHz.
The drop-and-dry method was used to disperse the nanomaterial suspension on the surface of the carbon-based screen-printed (SPE-C) sensors. A total of 10 μL of the suspension was poured onto the SPE-C surface, adding 5 μL at each step using an Eppendorf micropipette, thus constructing the single-layer carbon nanotube-based screen-printed (SPE-SWCNT) sensor. Solvent evaporation was carried out at room temperature in a desiccator.