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

Abstract

The widespread use of pesticides in the last decades and their accumulation into the environment gave rise to major environmental and human health concerns. To address this topic, the scientific community pointed out the need to develop methodologies to detect and measure the presence of pesticides in different matrices. Biosensors have been recently explored as fast, easy, and sensitive methods for direct organophosphate pesticides monitoring. Thus, the present work aimed at designing and testing a 3D printed adapter useful on different equipment, and a membrane support to immobilize the esterase-2 from Alicyclobacillus acidocaldarius (EST2) bioreceptor. The latter is labelled with the IAEDANS, a bright fluorescent probe. EST2 was selected since it shows a high specificity toward paraoxon. Our results showed good stability and replicability, with an increasing linear fluorescent intensity recorded from 15 to 150 pmol of labelled EST2. Linearity of data was also observed when using the immobilized labelled EST2 to detect increasing amounts of paraoxon, with a limit of detection (LOD) of 0.09 pmol. This LOD value reveals the high sensitivity of our membrane support when mounted on the 3D adapter, comparable to modern methods using robotic workstations. Notably, the use of an independent support significantly simplified the manipulation of the membrane during experimental procedures and enabled it to match the specificities of different systems. In sum, this work emphasizes the advantages of using 3D printed accessories adapted to respond to the newest research needs. Graphical abstract

A FRET Approach to Detect Paraoxon among Organophosphate Pesticides Using a Fluorescent Biosensor

“…p < 0.0001, Figure 2b) and high (Y = 12.57X + 98.1, R 2 = 0.99, F(1,3) = 2437, p < 0.0001, Figure 2c) protein amounts. Our results agree with previous results, where linear behaviour of the labelled EST2-S35C was obtained with different fluorescence-based methods [33].…”

“…A significant linear increase in the fluorescence intensity of IAEDANS after energy transfer from tryptophan was observed for both low (Y = 29.12X + 8.916, R 2 = 0.99, F (1,3) = 899.9, p < 0.0001, Figure 2b) and high (Y = 12.57X + 98.1, R 2 = 0.99, F (1,3) = 2437, p < 0.0001, Figure 2c) protein amounts. Our results agree with previous results, where linear behaviour of the labelled EST2-S35C was obtained with different fluorescence-based methods [33]. p < 0.0001, Figure 2b) and high (Y = 12.57X + 98.1, R 2 = 0.99, F(1,3) = 2437, p < 0.0001, Figure 2c) protein amounts.…”

“…In conclusion, this study paves the way to applying this bioreceptor for FRET measurements of real samples such as food and/or biological fluids, eventually combined with solid-state fluorescence measurements [33].…”

Monitoring of pesticide amount in water and drinkable food by a fluorescence‐based biosensor

“…To overcome this problem, the enzyme was immobilised on a membrane which was placed onto a support in a 3D adapter designed as part of the extracurricular activities developed during the 1‐year fellowship programme. The 3D support allows the washing of the membrane to remove unwanted substances such as pigments, improving the fluorescence measurements for food samples and avoiding fluorescence interferences in the aqueous solution (Rodrigues et al, 2021 ).…”

“…In the first part of the work programme, the fellow carried out the overexpression in mesophilic host Escherichia coli strain BL21 (DE3) of a mutant of the thermostable esterase‐2 (EST2) from Alicyclobacillus acidocaldariu s (Febbraio et al, 2011 ), to which the serine 35 was replaced by a cysteine residue (EST2‐S35C) near the catalytic site. EST2‐S35C was extracted and purified following a protocol already described in Carullo et al ( 2018 ), with slight modifications (Rodrigues et al, 2021 ). The microorganism was grown in an appropriate medium, the biomass was recovered by centrifugation and the protein was extracted by a sonication step.…”