Highly Sensitive Detection of Chemically Modified Thio-Organophosphates by an Enzymatic Biosensing Device: An Automated Robotic Approach

Abstract

Pesticides represent some of the most common man-made chemicals in the world. Despite their unquestionable utility in the agricultural field and in the prevention of pest infestation in public areas of cities, pesticides and their biotransformation products are toxic to the environment and hazardous to human health. Esterase-based biosensors represent a viable alternative to the expensive and time-consuming systems currently used for their detection. In this work, we used the esterase-2 from Alicyclobacillus acidocaldarius as bioreceptor for a biosensing device based on an automated robotic approach. Coupling the robotic system with a fluorescence inhibition assay, in only 30 s of enzymatic assay, we accomplished the detection limit of 10 pmol for 11 chemically oxidized thio-organophosphates in solution. In addition, we observed differences in the shape of the inhibition curves determined measuring the decrease of esterase-2 residual activity over time. These differences could be used for the characterization and identification of thio-organophosphate pesticides, leading to a pseudo fingerprinting for each of these compounds. This research represents a starting point to develop technologies for automated screening of toxic compounds in samples from industrial sectors, such as the food industry, and for environmental monitoring.

A 3D printable adapter for solid-state fluorescence measurements: the case of an immobilized enzymatic bioreceptor for organophosphate pesticides detection

“…These values are approximately one thousand times lower than previous detection levels obtained using the EST2labelled bioreceptor in aqueous solution [18]. On the other hand, a similar LOD was achieved for paraoxon using fluorogenic substrate in a robotic workstation [20,27].…”

“…EST2 specificity and sensitivity led to a calculated limit of detection (LOD) in the nanomolar range of paraoxon. As recently demonstrated, EST2 can also recognize other OPs [20,27]; however, the oxidation of phosphorothionate OPs by using a strong oxidant, such as N-bromosuccinimide, is needed prior to its covalent binding to the protein [27]. In fact, similarly to acetylcholinesterases, EST2 shows lower affinity toward phosphorothionate OPs, although it seems to be able to reversibly bind some of them, such as parathion and chlorpyrifos [18,19].…”

“…However, these systems present low stability after changes in environmental conditions and low specificity toward inhibitor compounds, possibly reacting with other compounds than OPs. In our previous works [19,20,26,27], esterase-2 from Alicyclobacillus acidocaldarius (EST2) was used as an alternative biosensor for OP detection. This enzyme shows a very high affinity toward paraoxon (SI Fig.…”

Use of an Artificial Miniaturized Enzyme in Hydrogen Peroxide Detection by Chemiluminescence

“…Process control and quality assessment of reclaimed water may be further improved by developing efficient multi-purpose devices [ 4 , 28 ]. In particular, different oxidants, other than hydrogen peroxide, such as chlorite and chlorine dioxide [ 70 ], may be revealed through the methodology developed here, and their consumption rate may be eventually coupled to the presence of several aromatic and organophosphate pollutants [ 71 , 72 , 73 ].…”

Point-of-Care Diagnostics of COVID-19: From Current Work to Future Perspectives

“…Nowadays, biosensors are already of common use, such as electrochemistry-based glucometers for glucose determination in blood [108,109] or lateral-flow-based devices for fertility and pregnancy tests [110,111]. Other biosensors have been proposed for several applications in healthcare, such as wearable biosensors for healthcare monitoring [112] for the detection of toxic substances in human fluids [113] or in foods [114], including possible real-time detection in the environment [115][116][117][118]. The simpler its use, the more a biosensor can be introduced into daily life with user-friendly self-explanatory interfaces.…”