Automatable Flow System for Paraoxon Detection with an Embedded Screen-Printed Electrode Tailored with Butyrylcholinesterase and Prussian Blue Nanoparticles

Abstract

Nowadays extensive volumes of pesticides are employed for agricultural and environmental practices, but they have negative effects on human health. The levels of pesticides are necessarily restricted by international regulatory agencies, thus rapid, cost-effective and in-field analysis of pesticides is an important issue. In the present work, we propose a butyrylcholinesterase (BChE)-based biosensor embedded in a flow system for organophosphorus pesticide detection. The BChE was immobilized by cross-linking on a screen-printed electrode modified with Prussian Blue Nanoparticles. The detection of paraoxon (an organophosphorus pesticide) was carried out evaluating its inhibitory effect on BChE, and quantifying the enzymatic hydrolysis of butyrylthiocholine before and after the exposure of the biosensor to paraoxon, by measuring the thiocholine product at a working voltage of +200 mV. The operating conditions of the flow system were optimized. A flow rate of 0.25 mL/min was exploited for inhibition steps, while a 0.12 mL/min flow rate was used for substrate measurement. A substrate concentration of 5 mM and an incubation time of 10 min allowed a detection limit of 1 ppb of paraoxon (corresponding to 10% inhibition). OPEN ACCESSChemosensors 2015, 3 130The stability of the probe in working conditions was investigated for at least eight measurements, and the storage stability was evaluated up to 60 days at room temperature in dry condition. The analytical system was then challenged in drinking, river and lake water samples. Matrix effect was minimized by using a dilution step (1:4 v/v) in flow analysis. This biosensor, embedded in a flow system, showed the possibility to detect paraoxon at ppb level using an automatable and cost-effective bioanalytical system.

An Overview of Recent Electroanalytical Applications Utilizing Screen‐Printed Electrodes Within Flow Systems

“…Organophosphorus pesticides were detected using SPEs coupled to FIA systems and modified with acetylcholinesterase or butyrylcholinesterase enzymes. [18,35,36] Different strategies were used for immobilization of the enzyme upon SPEs, such as the sequential placement of glutaraldehyde, bovine serum albumin, and Nafion on a SPE modified with Prussian blue NPs, [18] entrapment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 in a polymeric matrix of PVA (photo-polymerization between azide groups) on a SPE modified with cobalt(II) phthalocyanine [35] and through metal-chelate affinity and magnetic nanobeads. [36] In all the aforementioned papers, very low LODs (� 0.004 μmol L À 1 ) were obtained for the analysis of pesticides in natural water samples.…”

“…[81] Similar to FIA, BIA systems also allow for Gaussian shapes to be obtained via the injection of small solution volumes (10 to 150 μL). However, this is possible without the use of tubes and connections (without leaks or air bubbles), pumping systems, [13] Hydrogen peroxide Hair colorant and milk CNTSPE modified with metal NPs 20 450 [14] Biotin PF SPCE modified with horseradish peroxidase 0.008 13 [15] Paracetamol Naproxen PF SPCE and MWCNTSPE 0.4 0.3 90 [16] Methimazole Human serum and PF SPCE modified with (IrOxNPs) and tyrosinase 0.004 36 [17] Paraoxon Drinking, river and lake water SPGE modified with butyrylcholinesterase and PB NPs 0.004 NR [18] Gallic acid White and red wine SPEÀ Au-SAM/AuNPs-Linker/ Fullerenols/TvL 6.5 14 [34] Chlorpyriphos-oxon Malaoxon Milk SPCE modified with acetylcholinesterase 0.000005 19 [35] Organophosphate insecticide Lake water Carbon paste SPE modified with enzyme magnetic beads 0.003 18 [36] Nimodipine PF Carbon nanofiber SPE 0.08 NR [37] Pioglitazone PF SPE modified with MWCNT -polyvinyl chloride 0.8 90 [38] Oxicam derivatives PF, plasma and urine SPE modified with MWCNT -b-cyclodextrin 0.6 240 [39] [42] 3-hydroxyanthranilic acid Urine AuSPE modified with MIPs 0.02 225 [43]…”

“…Over recent years, many research groups have successfully demonstrated that homemade flowcells with different designs can be constructed and used for the positioning of SPEs. Flow-cells for FIA, [13][14][15][16][17][18] BIA, [19][20][21] and HPLC [22] systems are available in the literature. However, to attract potential new users, it is very important that a robust and easy to use flow systems for SPEs are commercially available.…”

Rapid electrochemical screening methods for food safety and quality

“…In a recent work we developed an amperometric biosensor for the determination of paraoxon, based on the enzyme butyrylcholinesterase (BChE) immobilized on screen-printed electrodes modified with Prussian Blue Nanoparticles (PBNPs), and embedded in a flow system [21].…”

Organophosphorous Pesticide Detection in Olive Oil by Using a Miniaturized, Easy-to-Use, and Cost-Effective Biosensor Combined with QuEChERS for Sample Clean-Up

“…Herein, we demonstrated the suitability of the well-known QuEChERS method as a sample treatment for complex food matrices in combination with a miniaturized, easy-to-use, and cost-effective electrochemical biosensor for paraoxon detection in olive oil, based on the enzyme butyrylcholinesterase (BChE) immobilized on carbon black nanomaterial (CB)-modified screen-printed electrodes (SPEs). The use of BChE as a bioreceptor for pesticide electrochemical monitoring has been widely documented in previous articles, where its high robustness and reliability were extensively demonstrated [ 8 , 9 ]. CB is likewise recognized as a cost-effective nanomaterial (only 1 euro for 1 kg of CB) to modify screen-printed electrodes, as it is able to enhance the electrochemical properties of the electrodes [ 10 , 11 , 12 , 13 , 14 , 15 , 16 ].…”