A novel automated flow-based biosensor for the determination of organophosphate pesticides in milk

This work describes the development of an automated flow-based biosensor that employs genetically modified acetylcholinesterase (AChE) enzymes B394, B4 and wild type B131. The biosensor was based on a screen printed carbon electrode (SPE) that was integrated into a flow cell. Enzymes were immobilised on cobalt (II) phthalocyanine (CoPC) modified electrodes by entrapment in a photocrosslinkable polymer (PVA-AWP). The automated flow-based biosensor was successfully used to quantify three organophosphate pesticides (OPs) in milk samples. The OPs used were chlorpyriphos-oxon (CPO), ethyl paraoxon (EPOx) and malaoxon (MOx). The total analysis time for the assay was less than 15 min. Initially, the biosensor performance was tested in phosphate buffer solution (PBS) using B394, B131 and B4 biosensors. The best detection limits were obtained with B394; therefore, this biosensor was used to produce calibration data in milk with three OPs in the concentration range of 5 × 10−6 M to 5 × 10−12 M. The limit of detection (LOD) obtained in milk for CPO, EPOx and MOx were 5 × 10−12 M, 5 × 10−9 M and 5 × 10−10 M, respectively, with a correlation coefficient R2 = 0.9910. The automated flow-based biosensor successfully quantified the OPs in different fat-containing milk samples. There were no false positives or false negatives observed for the analytical figures of merit for the constructed biosensors. This method is inexpensive, sensitive, portable, non-invasive and provides real-time results. This analytical system can provide rapid detection of highly toxic OPs in food matrices such as milk.

► Developed an automated flow based biosensor system which can control the dispensing of liquid (volumes) during the analysis of organophosphates.
► This is the first attempt of OPs analysis in milk using flow based automated system using highly sensitive genetically modified AChEs.
► This work demonstrates the sensitive determination of three priority compounds CPO, EPOx and MOx in milk.
► The LODs obtained for EPOx, CPO and MOx were 5 × 10−9, 5 × 10−12 and 5 × 10−10 M, respectively, with B394.