A paper-based biosensor was developed for the detection of the degradation products of organophosphorus pesticides. The biosensor quantifies acetylcholine esterase inhibitors in a fast, disposable, cheap, and accurate format. We specifically focused on the use of sugar or protein stabilizer to achieve a biosensor with long shelf-life. The new biosensor detected malathion with a detection limit of 2.5 ppm in 5 min incubation time. The operational stability was confirmed by testing 60 days storage at 4°C when glucose was used as stabilizer.
Detection of pesticide traces in food and water is an important safety issue due to intensive agricultural applications and their consequent toxicity. Pesticides, such as organophosphates (OP) and carbamates (CM), have inhibitory effects on cholinesterases which are enzymes essential for the proper functioning of the nervous system of vertebrates and insects. The toxic action of organophosphate and carbamates arise from the inhibition of acetylcholinesterase activity leading to accumulation of acetylcholine at the nerve endings and therefore causing cholinergic overstimulation characterized by severe consequences in humans including abdominal cramps, muscular tremor, hypotension, breathing difficulty, diarrhea, slowing heartbeat (bradycardia), muscular fasciculation, and paralysis [
The detection of pesticides or nerve agents has been traditionally carried out in laboratory settings with large and expensive instruments such as gas chromatography coupled with mass spectroscopy (GC-MS) [
Acetylcholine is a neurotransmitter active in central nervous systems and skeletal-muscle junction. Acetylcholine esterase (AChE) is the hydrolase that degrades acetylcholine molecules into choline and acetic acid, thus terminating impulse transmission at cholinergic synapsis. Therefore, AChE controls generation of nerve impulses in the postsynaptic neurons. Toxicity of OP and CM depends on inhibition of AChE; thus the enzyme is a common bioevaluator for the detection of organophosphates and carbamates [
In this study, a paper-based sensor was developed as a rapid and reliable monitoring method for organophosphates and carbamates. The model interaction between malathion and its specific inhibition of AChE was used to monitor quantitative changes in order to develop a mobile biosensor. Unlike the previously reported acetylcholinesterase-based paper biosensors, we evaluated sugar and protein stabilizers in order to develop a biosensor with improved shelf-life.
AChE (from electric eel), (5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), acetylthiocholine iodide (ATCh), and malathion were purchased from Sigma-Aldrich. The chemicals used in the preparation of buffers were purchased from Merck. Munktell No. 1 filter discs were purchased from Munktell (Falun, Sweden).
A mixture of enzyme, substrate, chromophore, and stabilizer solution was prepared for the indicated final concentrations in each experiment. For preparing biosensor strips, Munktell filter paper discs were prepared in 1 × 1 cm pieces and autoclaved at 120°C for 25 minutes prior to use (Figure
Schematic representation of biosensor support construction. (a) Munktell filter papers were cut and (b) fixed on a plastic support. (c) The enzyme mixture (AChE and DTNB) was directly applied on the fixed paper and dried. The samples with ATCh were directly applied on dried paper strips for color formation.
The ChE inhibition assay is based on quantification of free sulfhydryl groups of thiocholine which is the product of acetylthiocholine hydrolyzation by acetylcholine esterase. Ellman’s reagent, 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), is used to generate a yellow chromophore detectable at the 405 nm [
In this study, a paper-based sensor device was developed as a rapid and reliable monitoring method for OP and CM pesticides. The device is composed of dipstick paper sensor and a CCD camera for analysis in a portable format. The model interaction between malathion and its specific inhibition of AChE was used to monitor quantitative performance of the sensor. A biosensor for AChE inhibitory molecules has been developed by immobilizing the enzyme, its substrate (ATCh), and a chromophore (DTNB; 2-nitro-5-thiobenzoic acid) in paper matrix through adsorption. The chemical reactions leading to inhibitor dependent colour development are summarized in Figure
Two-step sequential reactions of acetylthiocholine (ATCh) for production of yellow colored TNB. In reaction 1, ATCh is broken to acetic acid and thiocholine (TCH). The free sulfhydryl group of TCh is quantified through Ellman’s method in reaction 2. The resulting TNB is used in a direct determination of the activity of ACh esterase.
To determine the working amounts of sensing components, a range of concentrations for each of them was systematically optimized for the best combination of AChE as the enzyme, DTNB as the chromophore, and ATChI as the artificial substrate. Optimum concentrations were selected according to the quantified colour production and the observation based on the naked eye, since occasionally the proposed biosensor would be used in the field with minimum instrumental help. Figure
The response of biosensor (a) at different substrate, (b) enzyme, or (c) Ellman’s reagent concentrations. In each experiment, ATCh, AChE, and DTNB were mixed and adsorbed into paper matrix. Their fixed concentrations were AChE 12 U/mL, DTNB 4
The effect of pH on the biosensor platform was investigated between 1 and 13 (Figure
(a) The response of biosensor at different pH values from 4 to 13. Red line (O) is the color intensity of DNTB and ATCh with AChE and black line (Δ) represents the color intensity without AChE. (b) Effect of temperature on biosensor response. Vertical bars indicate standard error of mean.
Thermal stability of AChE on paper was measured by an investigation at temperatures 4°C, 25°C, and 37°C. For all temperatures, the biosensors were incubated at that specific temperature for 5 minutes after the addition of ATChI solution to the mixture of other components. Temperature studies showed that at 4°C the activity of the enzyme ceased and no colour formation was observed. At room temperature and at 37°C, the yellow colour formation occurred with significantly different colour units with respect to control sensors.
Enzyme-based biosensors have been known for their selectivity, specificity, and catalytic signal amplification for the development of biosensors [
The stability of the biosensor was attempted to be improved by adding BSA into sensor mixture at concentrations up to 5% (w/v). There was about a 40% increase in colour development for 5% BSA addition (Figure
Effect of (a) BSA, (b) trehalose, (c) glucose, and (d) temperature on ChE activity. For (a), (b), and (c), black line is the color intensity for reaction mixture without enzyme, and red line is color intensity for reaction mixture with the enzyme.
Trehalose is a dimer of two glucose molecules and has recently been shown to help retain the functional stability of enzymes [
The shelf-life of biosensors were tested after they were incubated at 4 or 25°C in nontransparent plastic bottles in order to prevent direct light exposure because both DTNB and ATCh are light sensitive molecules. As seen in Figure
For evaluating the ability of developed sensor for quantifying AChE inhibitory effects, a commonly used inhibitor, malathion, was used by preparing a standard paper sensor with 4
Relationship between inhibition of AChE activity and malathion.
Performance of a dipstick-type acetylcholine esterase inhibitor sensor was investigated for determining optimal concentrations of sensor components and relevant enzyme stabilizers. The results in this study suggest that sugar stabilizers can be a potentially useful component in paper-based acetylcholine esterase inhibitor sensors. Among BSA, glucose, or trehalose, glucose was the best stabilizer in improving colour development and shelf-life at 4°C for especially visual tests. BSA improved the stability at higher levels compared to glucose, but the background levels did also increase to levels hindering visual quantification.