Sodium chloride (nacl)

The in vitro experiments required hemolymph collection. To collect the hemolymph, the larvae were selected according to the following criteria: last instar, weighing 240–300 mg, and presenting with clear, uniform color without dark spots or grayish marks. To collect the hemolymph, we held the larva in its ventral position, punctured one of the central prolegs and collected the hemolymph. Adipose tissue and any liquid of a dark color were discarded. The collected hemolymph of ten larvae was transferred to a microtube containing 900 μL of IPS (insect physiological saline: 150 mM sodium chloride (Promega, USA), 5 mM potassium chloride (Promega), 10 mM Tris HCl (Promega) pH 6.9, 10 mM EDTA (Promega) and 30 mM sodium citrate (Sigma-Aldrich, USA) plus 10 mM N-ethylmaleimide (Sigma-Aldrich) (anticoagulant). Tubes were placed on ice, and sample collection was carried out immediately to avoid cell melanization. After centrifugation at 2000 rpm and at 4 °C for 5 min, the supernatant was discarded, cells were washed with 500 μl of cold IPS and the contents of two tubes (or several, depending on the number of assays) were pooled together in a new tube. A second centrifugation was performed under the same conditions. The supernatant was again discarded, and the cells were resuspended in 1000 μl IPS. The number of cells was determined with a Neubauer chamber.

CM-dextran sodium salt (CMD-Na), phosphate-buffered saline (PBS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysulfosuccinimide (NHS), ethanolamine, potassium chloride (KCl), potassium ferricyanide (Fe(CN)₆³⁻), hydrogen peroxide (H₂O₂), diethyl pyrocarbonate (DEPC), and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich (Sigma-Aldrich St. Louis, MO, USA). Anti-fluorescein horseradish peroxidase (HRP) is a 40,000 dalton protein supplied by Abcam (Cambridge, UK). MES free acid monohydrate, hydroxymethyl-aminomethane (Tris), and ethylenediaminetetraacetic acid (EDTA) were purchased from Amresco (Amresco Inc., Solon, OH, USA). Core streptavidin was from BiVision (Bioptics, Tucson, AZ, USA). Sodium chloride (NaCl) was from Promega Corporation (Madison, WI, USA). The biotinylated ssDNA probe (bioreceptor probe) (5′-CAGACAGTGTTA-3′-(CH2)₆-biotin), fluorescein (FITC) ssDNA probe (detector probe) (FITC-(CH2)₆-5′-CCATCTTTAC-3′), and artificial mimic targeting sequence (5′-UAA CAC UGU CUG GUA AAG AUG G-3′) were designed by us and made by Genomics (Taipei, Taiwan). Screen-printed carbon electrodes (SPCEs) were purchased from Zensor R&D (Taichung, Taiwan).