# Electrochemical Detection of Penicillin G Using Molecularly Imprinted Conductive Co-Polymer Sensor
## 2 Materials and Methods
### Synthesis of the MICP  
As is often the case when we speak about a molecularly imprinted polymer, it is more precisely a copolymer as it includes a cross-linking monomer and a functional monomer. In this work, we used pyrrole (Py) and pyrrole-3-carboxylic acid (PyCOOH). The interest of this functional monomer is essentially its ability to create hydrogen bonds with other compounds, due to its carboxylic acid function, especially with other carboxylic acid functions. Figure 3: Substrates samples, from left to right, after printing: after gold deposit: after ink acetone removal (measurement scale in centimeters). The polymerization method used is oxidative polymerization. An electron is removed from the monomer, reacting with other monomers, and leading to the beginning of polymerization. Depending on the standard potential of the chosen oxidant, we will be in the presence of a more or less strong oxidation. However, it was observed [20] that the strength of the oxidant used will influence the crosslinking rate of the obtained polymer. It is obvious that this cross-linking will decrease the conductivity of the polymer. However, in the case of a MICP, this cross-linking will also make it possible to rigidify the structure and to ensure that a correct imprint is preserved after extraction of the target molecule. Hence, a strong oxidant will be used for the synthesis which is ammonium persulfate (APS). For all the syntheses, the molar ratios will be respected from one synthesis to the other. The aimed ratio in accordance with what has been observed in the literature for PenG is 12 molecules of Py and 4 molecules of PyCOOH for 1 molecule of PenG. This ratio will be regularly noted as 12/4 [21; 22]. The composition of the MICP polymerization solutions is the following: 8.97 \(\times\) 10\({}^{-4}\) mol/L of PenG, 3.555\(\times\)10\({}^{-3}\) mol/L of PyCOOH, 10\({}^{-2}\) mol/L of Py, 0.158 mol/L of H\({}_{2}\)SO\({}_{4}\), and 0.0355 mol/L of APS. The polymerization takes place for 20 min at 30 \({}^{\circ}\)C in 25 mL of water at pH 2 (adjusted with H\({}_{2}\)SO\({}_{4}\)). The APS is added about 15 min after all the other chemicals. This is the time needed to reach the working temperature, but it also allows molecules to correctly perform self-assembly before the beginning of polymerization. In order to increase the thickness of the sensitive layer, the polymerization is repeated twice in a row on the same substrate. The SO\({}_{4}\)\({}^{2-}\) ions come from H\({}_{2}\)SO\({}_{4}\) and especially from the reduction of APS, are used as dopants for our polymer and will allow us to stabilize the charges which will travel there. However, during this synthesis, the objective is not to realize the most conductive polymer. Indeed, with a polymer that is too conductive, especially at the core, it would be difficult to see a small variation in the conductivity of the material at the surface, when in contact with the target molecule. The ideal is therefore to have a conductivity just sufficient to ensure the charge transfer from one electrode to the other. The polymer is therefore not overly doped. The kinetics analysis of this oxidation has been performed at different temperatures in order to fully understand the polymerization mechanisms. Yet, the mechanisms of PPy polymerization are not unanimously accepted. The most known and the most widely accepted mechanism consists in the coupling of 2 radical cations resulting from 2 pyrrole monomer oxidation as presented in Figure 4a (resulting from a potential applied at an anode for the electropolymerization, or from the reaction with a chemical oxidizing agent, for chemical oxidation) [23]. Then the coupling may occur, forming bipyrrole, the dimer (cf. Figure 4c,_e_) [24]. This bipyrrole dimer is oxidized again and may couple with another monomer, dimer, or oligomer (cf. Figure 4f) to finally end up with a polypyrrole chain after the reaction between two cations (cf. Figure 4g). However, some more recent studies showed that the polymerization steps in water are a bit different.