Abstract
The development of faster, sensitive and real-time methods for detecting organophosphate (OP) pesticides is of utmost priority in the in situ monitoring of these widespread compounds. Research on enzyme-based biosensors is increasing, and a promising candidate as a bioreceptor is the thermostable enzyme esterase-2 from Alicyclobacillus acidocaldarius (EST2), with a lipase-like Ser–His–Asp catalytic triad with a high affinity for OPs. This study aimed to evaluate the applicability of Förster resonance energy transfer (FRET) as a sensitive and reliable method to quantify OPs at environmentally relevant concentrations. For this purpose, the previously developed IAEDANS-labelled EST2-S35C mutant was used, in which tryptophan and IAEDANS fluorophores are the donor and the acceptor, respectively. Fluorometric measurements showed linearity with increased EST2-S35C concentrations. No significant interference was observed in the FRET measurements due to changes in the pH of the medium or the addition of other organic components (glucose, ascorbic acid or yeast extract). Fluorescence quenching due to the presence of paraoxon was observed at concentrations as low as 2 nM, which are considered harmful for the ecosystem. These results pave the way for further experiments encompassing more complex matrices.
Highlights
Organophosphates (OPs) are a class of neurotoxic compounds including molecules with diverse applications from insecticides to herbicides or nerve agents [1]
The over-expression and biochemical characterisation of esterase-2 from Alicyclobacillus acidocaldarius (EST2)-S35C were described in detail in Carullo et al [23], confirming that the serine-to-cysteine residue mutation does not affect the catalytic site structure, and the structure–function relationship is unchanged
To EST2, EST2-S35C remains sensitive to paraoxon inhibition [23]
Summary
Organophosphates (OPs) are a class of neurotoxic compounds including molecules with diverse applications from insecticides to herbicides or nerve agents [1]. OPs’ high efficiency against target plagues and their low persistence has led to their wide use in urban and agricultural areas, and they are commonly found in environmental samples [2,3,4,5]. OPs act by inhibiting the activity of acetylcholinesterase (AChE) enzymes, leading to muscular dysfunction in target organisms [6]. Due to similarities in the AChE family among diverse groups of organisms, OPs pose a risk to non-target animals, including humans [7,8]. The widespread presence of OPs in the environment has led to a common effort to monitor their concentrations in environmental (water, soil, air and food) and human (urine, blood and tissue) samples
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