Homogeneous electrochemical sensing detection of organophosphorus based on hybrid lipase inhibition

Organophosphorus pesticides (OPs) have been widely employed in agricultural production due to their high efficacy and broad-spectrum activity. At the same time, their potent inhibition of acetylcholinesterase (AChE) can induce neurotoxicity and toxic effects, thereby posing a serious threat to food safety and environmental security. Consequently, much attention has been given to accurately monitoring the organophosphorus residues in food and water sources. Conventional detection of the organophosphates, such as gas chromatography, liquid chromatography, and mass spectrometry, can offer high sensitivity and accuracy. However, the expensive equipment and complex operation are typically required for the rapid on-site detection. A highly promising detection can be expected to combine the electrochemical biosensors with the high specificity of the enzyme-substrate interactions, due to the simplicity and cost-effectiveness of the electrochemical approach. Nevertheless, the conventional sensors on acetylcholinesterase or organophosphorus hydrolase can often suffer from low stability and high preparation costs. In this study, a homogeneous electrochemical sensing was developed to combine a hybrid lipase (BCL@Zn-hNF) with a gold nanoparticle-modified electrode (AuNPs/Au). The OPs were then detected to utilize the inhibition on the lipase to hydrolyse p-nitrophenyl palmitate. BCL@Zn-hNFs were prepared via self-assembly of Burkholderia cepacia lipase with Zn²⁺ ions. Three-dimensional flower-like nanostructures were obtained with a high specific surface area, catalytic activity, and peroxidase-like properties. The AuNPs were deposited onto a bare gold electrode via electrodeposition. Electrode assembly, AuNP size, and electrodeposition cycles were then optimized to enhance the electron transfer efficiency and the surface activity. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were employed to characterize the floral morphology of BCL@Zn-hNFs, while Fourier transform infrared spectroscopy (FT-IR) was employed to validate the coordination bond hybridization. Electrochemical impedance spectroscopy (EIS) further demonstrated that the electron transfer performance of the AuNPs/Au electrode was enhanced significantly after optimization. Results indicated that the AuNPs/Au modified electrode exhibited the optimal electron transfer efficiency (k_s = 1.15 s^-1) and effective surface area (0.23 cm²), when the AuNPs size was 14.18 nm and the number of electrodeposition cycles was 20. The high catalytic activity of BCL@Zn-hNF was obtained to protect the homogeneous system from enzyme conformation. The outstanding performance was achieved in detecting the methyl parathion (MP), indicating the broad linear range of 3.80×10^-8 – 4.56×10^-5 mol/L and a low detection limit of 1.13×10^-9 mol/L. The pH, temperature, and storage stability tests show that the residual activity of BCL@Zn-hNFs significantly outperformed the free lipase, thus retaining over 80% of its initial activity after 28 days of storage. Its tolerance was further validated in complex environments. Furthermore, the spiking tap water, vegetables, and fruit samples were used to verify the MP yielded recovery rates ranging from 100.37% to 134.62%, with RSD < 5%. Concurrently, this electrochemical sensing also demonstrated the excellent reproducibility (RSD = 1.91%), stability (retaining 90.96% activity after 30 days), and resistance to interference. In summary, this sensing platform can offer high sensitivity, high stability, and low cost, compared with conventional enzyme-based sensors. The finding can also provide a promising approach to detecting the organophosphorus pesticide residues. The enzyme-based electrochemical biosensors can be expected to extend into food safety and environmental monitoring.