A 50 mM (pH 9.5) Sodium tetraborate decahydrate buffer in water was prepared and used to make a 0.002 mmol APMA solution. For each fluorophore, 1 mg was weighed out in separate vials and suspended in 200 µL of the APMA solution. The mixtures were sonicated for 5 min before being incubated on an oscillator and room temperature for 24 h in the dark. The dyes were stored at −20 °C for further use.

For the synthesis of the nanosensors, 1.59 g AOT and 3.08 g Brij L4 were weighed out, mixed in a 250 mL round bottom flask and deoxygenated for 15–20 min using argon while being stirred. A 500 mL round bottom flask was used to deoxygenate 100 mL hexane for 30 min using argon before 42 mL of the deoxygenated hexane was added to the AOT and Brij solution. The flask was sealed with a stopper and a balloon under an argon atmosphere with continuous stirring. For the synthesis of neutral polyacrylamide nanosensors, 540 mg acrylamide and 160 mg bisacrylamide were dissolved in 1.5 mL deionised water, whereas 513 mg acrylamide, 152 mg bisacrylamide and 119 µL ACTA were used the preparation of positively charged polyacrylamide nanosensors. The APMA-fluorophore conjugates were added to the acrylamide solution using 15 µL OG-APMA, 15 µL FAM-APMA and 60 µL TAMRA-APMA. The mixture was deoxygenated and added to the flask containing the surfactants and hexane using a syringe. After 10 min, 30 µL of APS (10% w/v) and 15 µL TEMED were added. The flask was deoxygenated again, sealed, wrapped in aluminium foil and incubated on the stirrer for 2 h. After the incubation, the hexane was removed using a rotary evaporator at 30 °C and 30 mL of ethanol (100%) was added. The mixture was transferred to a 50 mL falcon tube and spun down at 3800 × g for 3 min. The supernatant was discarded, and the pellet re-suspended in 30 mL of ethanol (90% in water). The mixture was spun down again at 3800 × g for 3 min and washed twice in 30 mL ethanol (100%) and then suspended in 10 mL of ethanol (100%). The mixture was transferred into a clean 250 mL flask and the ethanol was removed using the rotary evaporator at 30 °C before the nanosensors were collected and stored at −20 °C in the dark until further use.

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