Elsevier

Environmental Research

Volume 174, July 2019, Pages 46-53
Environmental Research

Organophosphate hydrolase conjugated UiO-66-NH2 MOF based highly sensitive optical detection of methyl parathion

https://doi.org/10.1016/j.envres.2019.04.018Get rights and content

Highlights

  • OPH6His has been immobilized on a Zr-MOF, namely UiO-66-NH2.

  • 37% enhancement in the enzymatic activity is realized after immobilization.

  • Enzyme-MOF facilitates hydrolysis of methyl parathion in to p-nitrophenol (PNP).

  • PNP quenched the fluorescent of coumarin1 in proportion to pesticide content.

  • Detection of methyl parathion was made over 10–106 ng/mL.

Abstract

The hexahistidine-tagged organophosphorus hydrolase (OPH6His) has been immobilized on a Zr-MOF, namely UiO-66-NH2. The resulting enzyme-MOF composite was used as a carrier to facilitate the hydrolysis of an organophosphate pesticide, i.e., methyl parathion in to p-nitrophenol (PNP). The formation of PNP took place in direct proportion to the added pesticide concentration. Coumarin1 (7-diethylamino-4-methylcoumarin) was then introduced in the reaction mixture as a reporter fluorescent molecule. As PNP acted to quench the fluorescence of coumarin1, it became possible to detect methyl parathion over a wide concentration range of 10–106 ng/mL with an achievable limit of quantification as 10 ng/mL. The immobilization of OPH6His on the surface of UiO-66-NH2 was found to endow an improvement in the enzymatic activity by about 37%. The OPH6His/UiO-66-NH2 conjugate was reusable for at least up to eight times and also found stable toward long-term storage (minimum 60 days). The potential practical utility of the above proposed sensing method has been demonstrated by employing it for an accurate analysis of pesticide-spiked food samples.

Introduction

Organophosphorous pesticides (OPPs) constitute the most widely used (approximately 36%) class of pesticides (Lerro et al., 2015). Their overuse in recent decades has raised serious concerns about the safety of food and water supplies particularly in India. Since OPPs are known to inhibit the activity of acetylcholinesterase (AChE), their unwarranted exposure to human leads to serious health hazards related with the nervous, respiratory, digestive, and reproductive systems (Gupta, 2011) (Martin-Reina et al., 2017; Raanan et al., 2015; Sanchez-Santed et al., 2016). Therefore, it has become of paramount significance that a more routine monitoring of OPPs is carried out with the aid of rapid, portable and user-friendly methods. In this context, biosensors are envisaged as potential tools to serve the purpose.

The modern day biosensors mostly utilize advanced nanomaterials as efficient transducers which combine well with recognition biomolecules (e.g., enzymes, antibodies, whole cells and nucleic acids) (Kumar et al., 2015; Zhang et al., 2014). In context to the biosensing of pesticides, enzymes have been the most widely adopted recognition biomolecules due to their low cost, high efficiency and environment compliance (Agrawal and Rathore, 2014; Potara et al., 2018; Singh, 2008). A large number of these enzymatic biosensor works on the principle of AChE (acetyl cholinesterase) inhibition (Gong et al., 2009; Jiang et al., 2016; Li et al., 2018; Miao et al., 2016). The use of organophosphate hydrolase (OPH) for the catalytic biosensors has also been reported. For instance, a carbon nanotube (CNT)/OPH based electrochemical biosensor composed of a enzyme bilayer atop of the CNT film has been reported for the detection of methyl parathion with a detection limit of 0.8 μM (Deo et al., 2005). The use of cadmium telluride quantum dots (CdTe QDs) and gold nanoparticles (AuNPs) in combination with CNTs has also been suggested (Du et al., 2010). In a recent report, a conjugate of elastin-like polypeptide-organophosphate hydrolase, bovine serum albumin, titanium oxide nanofibers, and gold nanoparticles (ELP-OPH/BSA/TiO2NFs/AuNPs) has been advocated for highly sensitive detection of methyl parathion (Bao et al., 2017). The use of fluorescent reporter molecules, such as pyranine and coumarin has been reported to aid in the quantification of OPPs either directly or through detection of catalytic products (Orbulescu et al., 2006; Thakur et al., 2012).

Metal-organic frameworks (MOFs) are the crystalline porous materials which have recently been recommended for a variety of applications (e.g., adsorption, molecular separation, catalysis, drug delivery, imaging, and chemical sensing) with many beneficial features (Horcajada et al., 2010; Hu et al., 2014; Lee et al., 2009; Lei et al., 2014; Li et al., 2009). MOFs have also been reported as very efficient substrates for the immobilization of enzymatic proteins, such as horse radish peroxidise (HRP), bovine serum albumin (BSA), microperoxidase, and others (Jung et al., 2011; Liang et al., 2015; Mehta et al., 2016). Unique material properties of MOFs (e.g., high surface area, tuneable porosity, intrinsic or induced functionality, and possibility of post synthetic modifications) lend the MOF-enzyme composites with advantages of high protein loading, enhanced activity, improved stability, and biomolecule reusability (Jung et al., 2011; Liang et al., 2015; Lyu et al., 2014).

The present work, for the first time, reports the covalent immobilization of OPH6His (hexahistidine-tagged organophosphate) enzyme over a Zirconium-MOF (i.e., UiO-66-NH2). The resulting bioconjugate (OPH6His/UiO-66-NH2) has been investigated as a molecular carrier for the catalytic conversion of methyl parathion in to p-nitrophenol, which is then detectable with a simple addition of a fluorescent reporter, i.e. coumarin1. It is important to emphasize here that methyl parathion still remains one of the majorly used pesticides in developing countries like India despite a ban over its used by many government agencies (Bai et al., 2006; Gupta, 2004; Yadav et al., 2015). Many water, food and environmental samples are still found contaminated with toxic levels of methyl parathion (Krishna and Philip, 2008; Sousa et al., 2016; Yadav et al., 2015). Therefore, it is very important to develop portable and sensitive biosensing techniques which can help detecting the levels of methyl parathion in a rapid, convenient, and low-cost manner.

The OPH6His/UiO-66-NH2 bioconjugate has been synthesized by covalent binding of the enzyme over the surface of UiO-66-NH2 MOF, which was pre-activated with dicyclohexylcarbodiimide (DCC). Due to readily available single bondNH2 groups, UiO-66-NH2 facilitates a stable immobilization of enzyme. The selected MOF is also stable in aqueous conditions. As reported in some earlier works, UiO-66-NH2 can contribute in enhancing the activity of enzymes after their immobilization. The OPH6His/UiO-66-NH2 bioconjugate catalytically hydrolyzes methyl parathion into p-nitrophenol (PNP). The production of PNP has been monitored with the addition of a reporter dye molecule (7-isothiocyanato-4 methylcoumarin, or Coumarin1). The concentration of methyl parathion is correlated with the degree of fluorescence quenching. The proposed biosensing system has been demonstrated to work for both synthetic and real (spiked) samples. The herein reported OPH6His/UiO-66-NH2 bioconjugate has been proven to be a simple, specific, and highly efficient catalytic platform to facilitate the detection of an OPP, i.e., methyl parathion.

Section snippets

Chemicals

All the chemicals used in the study were of analytical grade purity. Zirconium chloride (ZrCl4), amino-terephthalic acid (NH2-BDC), N,N'-dicyclohexylcarbodiimide (DCC), 1,6-hexanediamine (HDA), dichloromethane (DCM), 2-[N-cyclohexylamino] ethanesulfonic acid (CHES), methyl parathion, coumarin1 (7-isothiocyanato-4 methylcoumarin), and dialysis membranes were procured from Sigma. Other chemicals including sodium phosphate (Na3PO4), sodium chloride (NaCl), nutrient broth, cobalt chloride (CoCl2),

Characterization of the purified OPH6His

The UV–Visible spectra of the purified OPH6His exhibited an absorbance peak at 280 nm typical to the presence of proteins (Fig. 2a). The peak is referred to indole and tyrosyl groups of aromatic amino acids such as tyrosine, tryptophan, and phenylalanine which are present in the primary structure of the enzymatic protein (Harm, 1980). The chromatographic purification of OPH6His was confirmed by SDS-PAGE experiment (Fig. 2b). A concentrated single band of 35 kDa appeared in different eluted

Conclusions

The present research work demonstrates that OPH6His enzyme can be efficiently conjugated with a functional MOF (i.e, UiO-66-NH2, containing surface single bondNH2 groups) via covalent immobilization. This approach provided a robust biointerfacing. The covalently immobilized OPH6His was characterized with almost 40% more enzyme activity compared to its free form. The enzyme-MOF composite has been used as the bioprobe in the co-presence of coumarin1 as a reporter molecule for sensitive and specific

Acknowledgements

Authors are thankful to the Director, CSIR-CSIO for providing the infrastructural facilities. The financial grant from Department of Biotechnology (DBT, India) project no. BT/PR18868/BCE/8/1370/2016 is gratefully acknowledged. JM is thankful to the University Grant Commission (UGC, India) for her research fellowship and SD thanks the Science and Engineering Research Board (SERB), Department of Science and Technology (DST, Govt. of India, New Delhi) for financial support (PDF/2016/002182).

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