Biossensor impedimétrico baseado em nanopartículas de CoFe2O4 acopladas a Concanavalina A para detecção de E. coli

Abstract

Rapid and accurate detection of pathogenic bacteria in point-of-care facilities, particularly in developing countries, is an emerging issue in food and medical industry. In this context, electrochemical and impedimetric biosensors have emerged as a powerful tool due to their low-cost, detection limits and sensitivity, where nanoparticles (NPs) can be used to modify electrodes to increase their surface area thus improving the sensitivity. This work presents the results obtained for a impedimetric biosensor built on a monolayer of cysteine (Cys) on gold containing NPs of CoFe2O4 and the lectin concanavalina A (ConA) aiming the detection of Escherichia coli (E. coli). Previously to the modification steps, the CoFe2O4 NPs were synthesized and functionalized with (3-aminopropyl)triethoxysilane (APTES) to provide NH2 ends (CoFe2O4@NH2). The cysteine molecules on gold were activated with N-hydroxysuccinimide (NHS) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) producing Au/Cys/NHS that, in turn, was immersed in an aqueous solution of CoFe2O4@NH2 forming Au/Cys/NPs. In the following step, this electrode was immersed in a solution containing Con A generating Au/Cys/NPs/ConA. Each modification step was monitored by electrochemical impedance spectroscopy and cyclic voltammetry in solution containing [Fe(CN)6]3-/4-. The values of charge transfer resistance (RCT) obtained from the Nyquist diagrams increased in each step varying from 50.43 to 532.56 Ω cm2 for the Au/Cys/NPs and Au/Cys/NPs/ConA, respectively, showing the surface blockage in respect to the [Fe(CN)6]3-/4- redox species after the lectin immobilization. The blockage effect was even stronger in solution containing E. coli and showed a concentration dependence on this bacterium. Such dependence allowed the use of the biosensor Au/Cys/NPs/ConA for detecting E. Coli within a linear range from 1.7 × 101 to 8.3 × 104 UFC/mL with an estimated limit of detection (LOD) of 0.2 × 10-3 UFC/mL after just 15 min of exposure. This low value of LOD was assigned to the combination of the nanometric properties of the CoFe2O4@NH2 NPs (electrical conductivity and high surface area) with the affinity of the ConA lectin for lipopolysaccharides on the cell wall of the E. coli bacteria. In comparison to other systems reported in the literature for gram-negative bacteria, the LOD value determined in this work indicates the production of an impedimetric biosensor fast (only 15 min of contact) and of low-cost and detection limit for the detection of E. coli.