# Carbon Nano-Onion Peroxidase Composite Biosensor for Electrochemical Detection of 2,4-D and 2,4,5-T
## 2 Materials and Methods
### Biosensor Preparation and Herbicide Detection  
The coating solution of the working electrode was prepared as follows: 0.5 mg of ox-CNO was mixed with 1 mg EDC and 2 mg NHS in 500 uL acetate buffer pH 5 and stirred at 4 \({}^{\circ}\)C for 1 h, followed by the addition of 2 mg of cystamine free base. The mixture was stirred for another 2 h at 4 \({}^{\circ}\)C, centrifuged and washed with water. The solid was resuspended in 500 uL of 0.1 M PBS pH 7.2 containing 0.5 mg NaClNH\({}_{3}\), sonicated for 5 min, and 1 mg of M-HRP was added. After 4 h at room temperature, the CNO/HRP conjugate was filtered through a 100 kDa Amicon(r) centrifugal filter to remove the excess of M-HRP, and the reentate (-160 uL) was mixed with 200 uL of a 10% ACDPSH suspension in PBS buffer pH 6.0. A clear solution was obtained after stirring overnight at room temperature, and 3 uL of this solution were dropped on the surface of the SPEs and dried under vacuum for 2 h (SPE/CD/CHP) electrodes.  
Control electrodes (SPE/CD/HRP) were prepared in a similar way but without CNOs. 0.5 mg of maleimide-activated HRP were mixed with 200 uL of 10% CDPSH suspension in PBS buffer pH 6.0 and stirred overnight at room temperature to obtain a clear solution. The activity of the conjugate was measured spectrophotometrically in order to ensure that both types of electrodes contained the same amount of immobilized HRP. Finally, 2.2 uL of this solution were dropped on the surface of the SPEs and dried under vacuum for 2 h (SPE/CD/HRP). The electrodes were stored in 20 mM PBS pH 7 in the fridge when not in use.  
Cyclic voltammetry (CV) and direct current (DC) amperometric measurements were carried out on an Autolab model PGSTAT 10 potentiostat/galvanostat controlled with GPES software (Eco Chemie, the Netherlands) in stirred electrolytes (425 rpm) at an applied potential of 0.0 V (vs. Ag/AgCl) in PBS pH 6. According to CV, at this potential, the electrochemical reduction of the enzyme-oxidized substrates takes place (see below). With the responses of the biosensors, the corresponding calibration curves were built by measuring the inhibition percent of HRP response in the presence of increasing concentrations of the herbicides. The SPE/CD/CNO/HRP biosensor was used to detect 2,4-D in soil. The soil sample (previously dried at 50 \({}^{\circ}\)C overnight) was homogenized manually, and 2,4-D was extracted by treating 1 g of solid with 2 \(\times\) 10 mL of methanol for 2 h. After centrifugation at 5000 rpm for 10 min, the methanol extract was isolated and evaporated, and the residue was dissolved in 2 mL of 0.1 mM NaOH to dissolve the herbicide. The pH was adjusted to 7 with HCl 6 M, and the aqueous sample was analyzed as described before. The extractions were performed in triplicate, and the results were compared with those obtained using an ELISA kit (Abnova) following the instructions of the manufacturer. 2,4,5-T was detected in spiked samples of water collected from the Francoli river (Tarragona city). The sample was collected at the coordinates 41\({}^{\circ}\)07\({}^{\prime\prime}\)12.0\({}^{\prime\prime}\) N+1\({}^{\circ}\)14\({}^{\prime}\)07.8\({}^{\prime\prime}\) E and filtered using a syringe filter (0.22 uL) before spiking with 3 uM 2,4,5-T.  
The stability of the biosensors stored in the fridge in PBS at 4 \({}^{\circ}\)C was studied for 40 days by measuring the inhibition of enzyme activity of 3 uM 2,4,5-T in 4-day intervals.  
Repeatability (repeated measurements on a single electrode) was studied on three identical electrodes until the signal decreased by 10% with respect to the original signal for the above concentration.