Acetylcholinesterase Biosensor Based on Paman Self-Assembled Monolayer-Modified Gold Electrode

The enzyme acetylcholinesterase, AChE, rapidly converts the neurotransmitter acetylcholine to choline and acetic acid after the transmission of a nerve impulse and the inhibition of this enzyme can lead to cholinergic dysfunction and death. The main responsible for AChE inhibition are the organophosphorous insecticides, which are the most used due to their high toxicity but low persistence in the environment. Therefore, the development of an AChE electrochemical biosensor in order to detect organophosphorous insecticides is of a great importance.  However, the immobilization of AChE on the electrochemical transducer without changing its structure is the most important challenge to obtain a sensitive biosensor. The immobilization by using self assembled monolayers (SAM) has been reported as a successfully alternative to fabricate biosensors. SAMs were usually prepared using the affinity of thiols such as alkanethiols for some metal surfaces, particularly gold. In this case, the main advantage consists in the immobilization of the enzyme close to the electrode surface with a high degree of control over the molecular architecture of the recognition interface, which provides a friendly environment for the enzyme. The objective of this research is to develop an AChE biosensor based on the enzyme immobilization onto a SAM modified gold electrode consisted of a dendrimer poly(amidoamine) (PAMAM) with a cystamine core. The modification of polycrystalline gold electrode was performed using monolayer of the PAMAM generation 4, with a concentration of 1.0 x 10^-2 mol L^-1. This molecule has in its nucleus one disulfide bond, that enables the direct chemisorption on the gold electrode, and it still has 64 terminals surface amino groups, which can anchor biomolecules, such as enzymes. In order to make the sulphur atom available for binding to Au electrode, NaHB₄ was used as a reducing agent. The immobilization of the AChE was evaluated by the immersion of the PAMAN-SAM-modified gold electrode in the enzyme solution using glutaraldehyde (GLU) as a crosslinking agent. The obtained AChE-PAMAN-SAM biosensor was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The measurements were carried out using an Autolab electrochemical system (Eco Chemie), with Ag/AgCl reference electrode, Pt wire as a counter and AChE-PAMAN-SAM as the working electrode. It was observed from the cyclic voltammograms obtained at different immersion times in PAMAN solution, that periods greater than 12 h showed no significant variation in the peak current intensity of the gold oxides of Au electrode, so this immersion time was chosen the further experiments. After the optimization related to the monolayer formation, this platform was used to immobilize the AChE enzyme. The enzyme immobilization was accompanied by the CV and EIS in the presence of the probe molecule, [Fe(CN)₆]^3-/4-, using two different methodologies: by dipping the PAMAN-SAM electrode in the AChE solution or by drop the enzyme onto the electrode. It was found that for both immobilization methods used, the current related to the [Fe(CN)₆]^3-/4- redox process decreased and the electron transfer resistance (Rct), estimated by diameter of the semicircle present at the high frequency region, increased, which is an indicative of the enzyme immobilization. However, the AChE-PAMAN-SAM obtained by the dip method showed higher oxidation current values in presence of enzyme substrate, acethylthiocoline (AChI). The construction of the biosensor was then optimized using different GLU and AChE concentration and a higher biosensor response was obtained using 1% v/v and 496 U/ml of GLU and AChE, respectively. A calibration curve was obtained for different substrate concentrations, enabling the determination of apparent Michaelis Menten constant Km= 1.62 x 10^-3 mol L^-1. Therefore, a substrate concentration of [AChI]= 2.00 x 10^-3 mol L^-1 was chosen for the inhibition measurements for the detection of pesticides.