Methyl parathio (MP) ( is an organophosphate (OP) pesticide, used as insecticide in agriculture to protect the crops from insects. Earlier, it was recommended as restricted pesticide for controlling the insect in the field of cotton, paddy, wheat, pulses such as green gram and black gram and oilseeds such as ground nut and mustard crops by Central Insecticide Board and Registration Committee (CIBRC). Food Safety and Standards Authority of India (FSSAI) has set MRLs of MP in food commodities in the range of 0.1-1 ppm. Although it was not recommended for fruits, vegetables and spices however many newspapers have reported the presence of this pesticide in vegetables and spices also. Recently in Aug 2018, Government of India had completely banned 18 pesticides including methyl parathion for the registrations, import, manufacture, formulation, transport, sell and any types of application. Therefore, there is an urgent need to develop a method that is rapid, sensitive, selective, reliable, economically feasible and able to monitor a large number of MP samples simultaneously in its MRL range.

Present prototype (Figure 1) is software controlled microplate based optical biosensor which is able to detect methyl parathion pesticide in the range of 0.1- 1.0ppm (MRL value). This prototype can be utilized for monitoring large numbers of sample (upto 96). It has two components: first is the biocomponent consisting of bio-hybrid immobilized on wells of microplate (96 wells) (Figure 1B) which hydrolyzes methyl parathion (transparent) into an optically detectable product (Figure 1A). Second is the microplate reader (optical transducer) which is completely controlled via software for all operations. This biosensor detects methyl parathion pesticide in sample in 5-10 min while traditional analytical techniques are not specific and require 3-5 days.

Methyl parathion (MP) is an organophosphate (OP) pesticide, used as insecticide in agriculture to protect crops from insects. In India, Central Insecticide Board and Registration Committee (CIBRC) has recommended methyl parathion as restricted pesticides in two different concentrations either in 2% DP or 50% EC for controlling insect pests in the field of cotton, paddy, wheat, pulses such as green gram and black gram and oilseeds such as ground nut and mustard crops. As per statistical reports by Directorate of PPQS, India, consumption of methyl parathion in India was 8408 MT during 2005 - 2010 and 5645 MT during 2010 - 2017. Methyl parathion causes inhibition of acetyl cholinesterase, which leads to excess accumulation of acetylcholine and causes overstimulation of muscle and nerve fibers, uncontrollable twitching, convulsions, difficulty in breathing or death. Thus, it has been classified by the World Health Organization (WHO) under ‘Category Ia’ (extremely toxic) and by the United States Environmental Protection Agency (US EPA) under ‘Toxicity Category I′ (most toxic) insecticide. EU/USDA/FSSAI (Food Safety and Standard Authority of India) had set the Maximum Residual Limit (MRL) of methyl parathion MP between 0.1 to 1.0 ppm depending on types of food commodities. Although methyl parathion was not recommended for fruits, vegetables and spices, however many newspapers have reported the presence of this pesticide in vegetables and spices probably because methyl parathion is cheaper than the other currently recommended pesticides. Recently in Aug 2018, Government of India has completely banned 18 pesticides including methyl parathion for the registrations, import, manufacture, formulation, transport, sell and any type of application. Therefore, there is an urgent need to develop a method that is rapid, sensitive, selective, reliable and economically feasible for monitoring of a large number of samples at very low concentrations.

Earlier BARC had developed handheld colorimetric biosensor (Technology Code AB28NABTD) that detects methyl parathion pesticide in the range of 1 - 10ppm. However, FSSAI has set MRLs of methyl parathion in food commodities in the range of 0.1- 1 ppm. Therefore, there is need to develop a method that is more sensitive and able to monitor a large number of samples simultaneously in its MRL range.

Present prototype is software controlled microplate based optical biosensor which is able to detect methyl parathion pesticide in the range of 0.1- 1.0ppm (MRL value). This prototype can be utilized for monitoring large numbers of sample (upto 96). It has two components: first is the biocomponent consisting of bio-hybrid immobilized on wells of microplate (96 wells) (Figure 1) which hydrolyzes methyl parathion (transparent) into an optically detectable product (Figure 2). Second is the microplate reader (optical transducer) which is completely controlled via software for all operations including data reduction, monitoring and analysis of each well of 96 wells microplate (Figure 3). A 200 µL volume of methyl parathion sample is added into the wells where the bio-hybrid has been immobilized and readings are acquired. This microplate based optical biosensor is initially calibrated with PNP, followed by methyl parathion in association with immobilized biocomponent. This biosensor specifically detects methyl parathion pesticide in sample in 5-10 min while traditional analytical techniques are not specific and require 3-5 days.

Salient feature

• Room temperature operation
• Multiple sample analysis (96 samples)
• Less analysis time (5-10 min)

Specifications

The biosensor showed a linear detection range from 0.1 to 1 ppm of methyl parathion pesticide. Response time (~95%) of microplate reader for analysis is typically ~ 2-3 min. Biosensor is operated at room temperature.

Applications

This biosensor can be used for specifically detecting methyl parathion pesticide in large numbers of samples in the range of MRL value.

Advantages

• This biosensor is specific for detecting methyl parathion pesticide in sample.
• Simultaneous analysis of multiple samples (96 samples).
• Detection of methyl parathion pesticide in the range of MRL value.
• Less analysis time (5-10 min) than conventional analytical techniques (3-5 days).

Equipments

• Autoclave
• Laminar flow
• Refrigerator (optional)
• Common glass wares

Requirements

• Raw Materials: Microplate, Silica nanoparticles, PEI, Sphingomonas sp. culture, Wakimoto media
• EquipmentsIncubator shaker, laminar flow, centrifuge, refrigerator, microplate reader
• Space requirements:The space requirement is approximately 15ft by 15ft to comfortably accommodate the process equipments such as incubator shaker, laminar flow, centrifuge, refrigerator, microplate reader etc.
• Man power:One technician to incubate the microbial culture, grow the cells, harvest the cells and access the hydrolytic activity for methyl parathion followed by immobilization on microplate and one scientist to carry out hydrolytic activity, calibration, validation and data analysis which is acquired by microplate reader.

            

Fig. 1 : Hydrolysis reaction of Methyl parathion pesticide with immobilized bio-hybrid                                Fig. 2 : Bio-hybrid immobilized on wells of Microplate (96 wells)

Fig. 3 : Microplate reader with optical transducer