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

Environmental Research 174 (2019) 46–53

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

T

Jyotsana Mehtaa,b, Sarita Dhakaa, Ashok K. Paulc, Siddavattam Dayanandad, Akash Deepa,b,∗ a

Nanoscience and Nanotechnology Lab (Division: H-1), Central Scientific Instruments Organisation (CSIR-CSIO), Sector 30C, Chandigarh, 160030, India Academy of Scientific and Innovative Research (AcSIR-CSIO), Sector 30C, Chandigarh, 160030, India c Desh Bhagat University, Mandi Gobindgarh, Punjab, India d School of Life Sciences, University of Hyderabad, Hyderabad, 500046, India b

A R T I C LE I N FO

A B S T R A C T

Keywords: Zr-MOF Organophosphate hydrolase Bioconjugate Enzyme activity Pesticide Biosensing

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 longterm 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.

1. 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

∗ Corresponding author. Nanoscience and Nanotechnology Lab (Division: H-1), Central Scientific Instruments Organisation (CSIR-CSIO), Sector 30C, Chandigarh, 160030, India. E-mail address: [email protected] (A. Deep).

https://doi.org/10.1016/j.envres.2019.04.018 Received 6 December 2018; Received in revised form 16 April 2019; Accepted 18 April 2019 Available online 19 April 2019 0013-9351/ © 2019 Elsevier Inc. All rights reserved.

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2.2. Instruments

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 eNH2 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.

Ultraviolet–visible (UV–Vis) absorbance, infrared (FTIR) transmittance, and photoluminescence (PL) studies were performed using UV–Vis–NIR (Varian Cary 5000, Agilent), Fourier transform infrared (FTIR) (Nicolet iS10, Thermo Fischer Scientific) and Photoluminescence (Varian Cary, Eclipse, Agilent) spectrophotometers, respectively. A thermogravimetric analyzer (Mettler Toledo) was used for the thermogravimetric analysis (TGA). The structural, morphological, and crystallographic characterizations were carried out with field emission scanning electron microscope (FE-SEM, S4300/SE, Hitachi), transmission electron microscope (TEM, Tecnai, G2-F20, 200 keV, FEI) and X-ray diffractometer (XRD, Bruker, D8 Advance). The surface area of the synthesized materials was analyzed using a BELSORP-max system from Microtrac. The affinity chromatography was run with a NiSepharose column fitted to the AKTA (Basic) FPLC system (GE Health Care). High-performance liquid chromatography based analysis were run on a system from Waters, equipped with a Symmetry C18 column (150 mm × 4.6 mm, 5 μ, Thermo Hypersil, Thermo Fischer Scientific, Waltham, MA, USA) and a UV–Vis detector (Waters 2487, Milford, MA, USA) set at 278 nm. We used a mixture of acetonitrile (HPLC grade) and Milli-Q water (40/60, v/v) as mobile phase (flow rate = 0.5 mL/min).

2.3. Purification and characterization of six-histidine tagged organophosphate hydrolase (OPH6His) enzyme The method for the expression and purification of OPH6His was adopted from the literature (Gorla et al., 2009; Pandey et al., 2009). After expression of OPH6His from E. coli BL21 (pSM5) cells, the desired enzyme was purified with an affinity chromatography column as described in an earlier published article (Kanugula et al., 2011). After purification, different eluted portions were pooled and then dialyzed against a solution of 50 mM Na3PO4 buffer containing 50 μM CoCl2 and 2% glycerol (pH 8.0). The dialyzed protein was characterized both qualitatively and quantitatively with UV–Vis absorption spectroscopy and polyacrylamide gel electrophoresis (12% SDS-PAGE). The enzyme activity of OPH6His was estimated by correlating its efficiency toward the conversion of methyl parathion (MP) into p-nitrophenol (PNP) (Fig. 1). For this, an aliquot of the purified OPH6His was mixed with 200 mM CHES buffer which also contained 200 μM of methyl parathion and 10 μM of CoCl2 in a 1:9 v/v ratio. The contents were incubated at 37 °C for 5 min which led to the formation of PNP. The absorbance of the resulting solution was measured at a wavelength of 410 nm. The specific activity of the purified OPH6His enzyme was then calculated according to the equation (1) (Dumas et al., 1989):

Specific activity = 2. Materials and methods

A ε× L× T× Mo

(1)

where A = absorbance; ε = extinction coefficient of PNP at 410 nm (M−1cm−1); L = Path length (cm); T = time (minutes); and M˳ = sample quantity (mg).

2.1. 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), and imidazole were purchased from Himedia. The Ni-Sepharose column matrix was purchased from Merck, India. The organophosphate hydrolase (OPH6His) enzyme was expressed as cloned opd gene in recombinant Escherichia coli bacterial cells. It was then purified following the affinity chromatography based method.

2.4. Synthesis of Zirconium-MOF (UiO-66-NH2) UiO-66-NH2 MOF was synthesized using a solvothermal-assisted reaction (Katz et al., 2013). Briefly, 2.8 mmol of ligand (NH2-BDC) were first dissolved in 20 mL of dimethylformamide (DMF) by ultrasonication for 20 min. Separately, 2 mmol of ZrCl4 were dissolved in18 mL of a solvent mixture (i.e., HCl:DMF, 1:5 v/v) by ultrasonication (20 min). The above prepared ligand and metal salt solutions were mixed and left to react overnight in a Teflon lined autoclave (T = 80 °C). Finally, the product (UiO-66-NH2) was collected, washed with DMF and ethanol (three-times each), and vacuum dried for 3 h at 90 °C. 47

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Fig. 1. Reaction mechanism of the OPH6His catalysed degradation of methyl parathion.

Specifically, the PL intensity of 460 nm fluorescence peak was monitored. In each of the case, the value of relative fluorescence intensity (RFI) was calculated as follows:

2.5. Surface immobilization of OPH6His on UiO-66-NH2 OPH6His has been immobilized on the surface of UiO-66-NH2 MOF. For this, the pre-synthesized UiO-66-NH2 MOF was first chemically activated employing the N,N′-dicyclohexylcarbodiimide (DCC)-mediator based chemistry (Shih et al., 2012). Briefly, 10 mg of UiO-66-NH2 was suspended in 1 mL mixture of DCC (1% w/v) and hexamethylenediamine (HDA, 1% w/v) taking dichloromethane (DCM) as solvent. The contents were stirred for 4 h at 10 °C. The activated MOF was then recovered from the reaction mixture by centrifugation (7000 rpm, 20 min), followed by washings with 200 mM CHES buffer, acetone, and ice-cold water. Finally, the activated MOF powder was dried overnight under vacuum. In the next stage, the activated UiO-66-NH2 was suspended in 1000 μL of CHES buffer (200 mM, pH 8.0) followed by the addition of 1000 μL of OPH6His solution (2 mg/mL). The reaction mixture was left to incubate for 1 h at 4 °C. The resulting product (i.e., OPH6His/UiO-66-NH2) was purified by providing a washing step with CHES buffer. The desired bioconjugate, i.e. OPH6His/UiO-66-NH2, was then dried in vacuum at room temperature. Supernatant, collected after the washing step, was used for the indirect estimation of the amount of enzyme immobilized over the MOF surface. For this, a standard BCA assay was performed (Smith et al., 1985), while calculations were made according to the equation (2).

RFI =

FIo − FIn where , n = 1,2,3…..n FIo

(3)

where FIn and FIo = fluorescence intensities of the reporter molecule in the presence and absence of methyl parathion, respectively. The performance of OPH6His/UiO-66-NH2 bioconjugate toward the catalysis of methyl parathion was evaluated in terms of several important parameters, such as specificity, regeneration, storage stability, and real sample analysis. The specificity was analyzed with respect to some other commonly used pesticides, such as dichlorodiphenyltrichloroethane (DDT), carbamate mix, and atrazine. The regeneration of the used OPH6His/UiO-66-NH2 was performed by recovering it from the reaction mixture by centrifugation (12,000 rpm, 10 min). The recovered material was then washed thrice with CHES buffer before overnight drying in vacuum at room temperature (RT, 25 ± 2 °C). The shelf-life of the bioconjugate was investigated as it was stored for prolonged durations in refrigerated conditions (4 °C). 2.7. Preparation of pesticide spiked food samples In order to evaluate the practical utility of the developed method, some food samples (orange and tomatoes) were analyzed after spiking them with known concentrations of methyl parathion. Properly washed oranges and tomatoes were first chopped and homogenized. 100 g of each of the homogenized sample was then dissolved in 50 mL of PBS by stirring for 1 h at RT. After filtration, the obtained filtrate was further purified from the suspended particles by centrifugation (8000 rpm, 10 min). The collected supernatant was then spiked with known concentrations of methyl parathion. The quantification of the pesticide content was also verified with the HPLC measurements.

Amount of enzyme immobilized onto the MOF surface = Initial amount of enzyme taken for immobilization − Amount of enzyme left in the supernatant after immobilization (2) 6His

The enzyme activity of the OPH /UiO-66-NH2 sample was determined by following a protocol as already described in section 2.3. 2.6. Application of OPH6His/UiO66-NH2 for detection of methyl parathion

3. Results and discussion

Methyl parathion is hydrolyzed by the OPH enzyme into a chromogenic product, p-nitrophenol (PNP). We have used a fluorescent reporter molecule (coumarin1 (7-isothiocyanato-4 methylcoumarin)) whose emission is quenched in proportion to the amount of PNP formed during the above reaction. Different dilutions of methyl parathion (10–106 ng/mL) were prepared in a mixed solvent of 200 mM CHES and 10 μM CoCl2. To each of the above samples (900 μL), 100 μL of the bioprobe solution (10 mg/mL OPH6His/UiO-66-NH2) was added. These mixtures were incubated at 37 °C for 5 min resulting in the formation of PNP. Subsequent centrifugation (12,000 rpm for 5 min) and membrane filtration (0.1 μM polytetrafluoroethylene (PTFE)) steps allowed the separation of the enzyme powder (i.e., used OPH6His/UiO-66-NH2) and the formed product (i.e., PNP as filtrate). A 100 ppm of coumarin1 (in 95% ethanol) was then added into the collected fractions of the above filtrates. After allowing the quenching reaction to complete (30 s), the fluorescence of resulting solutions was measured with a PL spectrophotometer.

3.1. 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 SDSPAGE experiment (Fig. 2b). A concentrated single band of 35 kDa appeared in different eluted fractions (lane 2–6). This observation clearly implies that the pure enzyme was recovered in a significant concentration after the chromatographic step. The activity of the enzyme (in terms of specific activity) was determined employing a previously reported method (Dumas et al., 1989). Its value has been estimated 295 IU/mg. 48

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Fig. 2. (a) UV–Visible spectra of purified OPH6His; (b) SDS-PAGE photograph, Lane 1- Protein molecular weight standard marker, Lanes 2 to 6 - Eluted fractions of the purified OPH (35 kDa).

CeN stretching near 1615 and 1150-1300 cm−1, respectively. The presence of CeH stretch bands around 2800-2900 cm−1 has further helped in validating the successful immobilization of OPH6His on the surface of UiO-66-NH2.

3.2. Characterization of UiO-66-NH2 and OPH6His/UiO-66-NH2 3.2.1. Spectrophotometric characterizations The UV–Visible absorption spectra of UiO-66-NH2 and OPH6His/ UiO-66-NH2 are shown in Fig. 3a. UiO-66-NH2 exhibited two characteristic absorption peaks: (i) at 270 nm, and (ii) broad band around 365 nm. The first peak can be attributed to the ligand-to-metal charge transfer (LMCT), while the second peak is related with the interaction of the lone pair of electrons of amino groups with the π* orbital of benzene ring (Nasalevich et al., 2014; Shen et al., 2013). The sample of OPH6His/UiO-66-NH2 showed an additional protein specific band at 280 nm to indicate a successful attachment of OPH6His enzyme with MOF. FTIR investigations for both UiO-66-NH2 and OPH6His/UiO-66-NH2 were performed in ATR mode (Fig. 3b). The sample of UiO-66-NH2 showed bands for carboxylic group vibrations at around 1490–1590 and 1390 cm−1 (Cai et al., 2013). The signals at 3477 and 3367 cm−1 were indicative of asymmetric and symmetric vibrations of the substituted amine group, respectively (Shen et al., 2013). A band centered at 1624 cm−1 could be attributed to the NeH bending whereas the signal around 1246 cm−1 corresponded to the CeN stretching of the aromatic amines (Kandiah et al., 2010). The immobilization of OPH6His enzyme onto the surface of UiO-66-NH2 took place via the formation of amide bond between the carboxylic group of the activated MOF and amine group of the enzyme (Shih et al., 2012). Further, the hydrogen bonds formed between amine functionality of MOF and carboxyl moiety of enzyme should also be attributed to a highly stable immobilization (Shih et al., 2012). The FTIR investigations provided the evidence of the above bonding via signals of amide bond absorption and

3.3.2. Structural investigations TEM (Fig. 4) and SEM (Fig. S1) images of the UiO-66-NH2 and OPH6His/UiO-66-NH2 samples revealed the formation of isoreticular crystals (Gomes Silva et al., 2010). These analyses also provided a clear indication about an intact crystallinity of the MOF even after the decoration of enzyme over its surface. XRD patterns (collected at a scan rate of 10 s/step) also supported the above observation. As Fig. 4c shows, both the samples of UiO-66-NH2 and OPH6His/UiO-66-NH2 were characterized with almost unchanged diffraction peaks. The collected XRD patterns also matched well with the simulated pattern as shown in Fig. 4c. A similar diffraction pattern for the crystalline UiO-66-NH2 has been reported in earlier studies. (Shen et al., 2013). UiO-66-NH2 consists an inner core of Zr6O4(OH)4 in which μ3-O and μ3-OH groups alternatively cap the Zr6-octahedron. BET surface area and porosity of the UiO-66-NH2 and OPH6His/UiO66-NH2 samples were investigated by recording N2-sorption isotherms at 77 K (Fig. 4d). The values of BET surface area were found to be 765 and 453 m2/g for UiO-66-NH2 and OPH6His/UiO-66-NH2, respectively. The value of BET surface area obtained for UiO-66-NH2 correlated well with the literature (Long et al., 2012). A decrease in the surface area after the immobilization of the enzyme should also be taken as an indicator to verify a successful immobilization of OPH6His onto the surface of UiO-66-NH2. The TGA profiles of the UiO-66-NH2 and OPH6His/UiO-66-NH2

Fig. 3. UV–Visible absorbance (a) and FTIR spectra (b) of OPH6His, UiO-66-NH2, and OPH6His/UiO-66-NH2. 49

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Fig. 4. (a) TEM image of UiO-66-NH2; (b) TEM image of OPH6His/UiO-66-NH2; (c) XRD patterns of UiO-66-NH2 (simulated and experimental) and OPH6His/UiO-66NH2; and (d) N2 adsorption isotherms for UiO-66-NH2and OPH6His/UiO-66-NH2.

66-NH2 bioconjugate possesses nearly 37% more specific activity (403 IU/mg) than the bare OPH6His (294 IU/mg). This significant enhancement in the activity can be attributed to the fact that the covalent immobilization of enzyme onto the surface of MOF provides structural stability to the enzyme. As an immobilization matrix, MOF has provided more surface area for the attachment of OPH6His particles. Due to the high loading of enzyme, the MOF-OPH conjugate is characterized with improved enzymatic activity. The immobilization of enzyme helps to maintain the orientation of the active site more frequently towards the substrate, thereby increasing the chances of collision between the active site and the substrate. Some preliminary studies revealed that due to its porous nature UiO-66-NH2 can also act as an adsorbent for methyl parathion (Fig. S5). Therefore, it provided a kind of pre-concentration of methyl parathion which in turn is useful to ensure better interaction of the pesticide with immobilized enzyme. Hence, the application of UiO-66-NH2 as a carrier of OPH6His is likely to aid the enzymatic catalysis of methyl parathion on multiple accounts. It is also important to mention here, no catalytic product (for example PNP) was found to be formed during the adsorption tests of methyl parathion on bare UiO-66-NH2.

samples were recorded to determine their thermal stability (Fig. S2). In case of UiO-66-NH2, the sample showed weight loss of any significant degree only beyond 300 °C which could be associated to the volatilization of linker molecules (Katz et al., 2013). In the sample of OPH6His/ UiO-66-NH2, the first stage of decomposition was observed at a relatively lower temperature (100 °C) due to the presence of additional enzyme content. However, it must be noted that even this degree of thermal stability is a good indicator that the herein proposed biomaterial (enzyme-MOF) should find extended applications in commercially viable biocatalytic processes. It may be pointed out here that in their solubilized form, enzymes can generally function only at temperatures below 40 °C. 3.3.3. Quantification of enzyme quantity immobilized on the surface of UiO-66-NH2 MOF and determination of activity of OPH6His/UiO-66-NH2 Employing BCA assay for protein estimation, the amount of OPH6His immobilized onto the surface of UiO-66-NH2has been calculated to be 1.25 mg of enzyme per 10 mg of MOF (equation (2), Fig. S3). Further, the enzymatic activity of the OPH6His/UiO-66-NH2 bioconjugate was evaluated as per the steps described in Section 2.3. A 100 μL sample of OPH6His/UiO-66-NH2 (10 mg/mL stock containing 1.25 mg of immobilized OPH6His) has been found to exhibit a specific activity equalling to that of a 100 μL of 2 mg/mL stock of the purified OPH6His (Fig. S4). Thus, the above correlation revealed that the OPH6His/UiO-

Fig. 5. Detection of methyl parathion using OPH6His/ UiO-66-NH2. (a) PL spectra of samples (free coumarin, coumarin + OPH6His/UiO-66-NH2, coumarin + OPH6His/UiO-66-NH2+ methyl parathion (0.05–106 ng/mL)); (b) Logarithmic calibration plot to depict the variation of RFI (measured at 460 nm) with different concentrations of methyl parathion.

50

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3.4. Detection of methyl parathion using OPH6His/UiO-66-NH2 bioprobe

UiO-66-NH2 has now been estimated with HPLC experiments. The results of the study are presented in Table S1. The action of enzyme-MOF converted 90% of methyl parathion in to p-nitrophenol (PNP) and dimethyl thiophosphate over a period of 20 h. No further degradation was possible. However, it must be noted here that the present sensing approach did not essentially require an extended conversion of methyl parathion in to PNP. Only 5 min of reaction is sufficient to allow the formation of PNP, which could quench the emission of coumarin. The regeneration studies were also conducted. These studies have indicated that the OPH6His/UiO-66-NH2 bioprobe can retains its activity to catalyze the formation of PNP for at least up to 4 regeneration cycles. During these cycles, the recycled bioprobe showed good response stability (about 10% decline only, Fig. 6a). This level of regeneration capacity of the enzymatic bioprobe is a good indicator toward its practical viability. A covalent immobilization of the enzyme over the surface of MOF prevented its leach-out during the regeneration steps. Further, studies were extended to assess the long-term stability of the above bioconjugate when it was stored in a dried form for 60 days (4 °C). As the data shown in Fig. 6b highlighted, the OPH6His/UiO-66NH2 bioconjugate could retain almost 80% of its response stability even after 60 days of shelf storage. The covalent immobilization of enzyme prevented the distortion of protein structure which contributed toward its enhanced stability during long duration storage. The inter-assay precision of the OPH6His/UiO-66-NH2 system has also been assessed. This was done by taking four different sensor electrodes, prepared under identical conditions, and using them separately to analyze a fixed analyte concentration (50 μg/mL methyl parathion). These electrodes’ output values (i.e., RFIs) displayed a coefficient of variance of 5% to highlight about a fair level of reproducibility.

3.4.1. Spectrophotometric detection The OPH6His/UiO-66-NH2 bioconjugate was incubated against varying concentration of methyl parathion ranging from 10 to 106 ng/ mL (Fig. 5). After necessary separation and purification steps, a suitable volume of coumarin1 was added in to the reaction filtrate containing the PNP product which formed as a result of interaction between the immobilized enzyme and the pesticide (Section 2.6). The fluorescence intensity of the untethered reporter coumarin1 (emission peak at 460 nm, excitation at 360 nm) was measured. As depicted in Fig. 5a, the degree of quenching of the fluorescent intensity was directly proportional to the amount of methyl parathion added in the reaction solution. The values of relative fluorescence intensity (RFI) for each of the measurement was calculated according to the equation (3). The value of FIo was estimated for a reaction mixture containing blank OPH6His/UiO66-NH2 (1 mg/mL, without any pesticide content) and coumarin1. A logarithmic representation of the calibration plot between RFI and pesticide concentration is shown in Fig. 5b. It is observed that the proposed biosensing system can offer a sensitive detection of methyl parathion over a broad dynamic detection range, i.e., from 10 to 106 ng/mL. The value of actually achievable detection limit for the present OPH6His/UiO-66-NH2 based detection system is 10 ng/mL or 38 nM, As such, this detection limit is comparable or better than most of the previously reported spectrophotometry based enzymatic detection of methyl parathion (Table 1). Importantly, our system is capable to offer the detection of methyl parathion over a wide range of concentration which is highly desirable for any practically viable sensor. During the process of detection, OPH6His/UiO-66-NH2 first catalysed the formation of PNP. Since the absorption band of PNP overlapped with the emission band of coumarin1, PNP functioned as an acceptor molecule to quench the emission energy of coumarin via fluorescence resonance energy transfer (FRET) mechanism. As a result, a quantifiable decline in the emission intensity of system in direct proportion to the concentration of pesticide was observed. The structural stability of OPH6His/UiO-66-NH2 after the detection of methyl parathion has been confirmed with SEM analysis. Fig. S1c provides useful information that OPH6His/UiO-66-NH2 remain structurally stable after its recovery from the reaction mixture.

3.4.3. Specificity of OPH6His/UiO-66-NH2 bioprobe and analysis of spiked real samples The specificity of the OPH6His/UiO-66-NH2 bioconjugate for methyl parathion was tested in presence of some non-specific (non-organophosphate) pesticides, such as DDT, carbamate mix, and atrazine (Fig. 6c). As the results clearly suggested, the OPH6His/UiO-66-NH2 bioprobe did not support the degradation of any of the above nonspecific pesticides into any product. There was insignificant quenching in the fluorescence in case of all the non-specific pesticides. Hence, it can be claimed that the present bioconjugate possesses a high specificity toward methyl parathion. Similarly, the bioconjugate can also be utilized for other organophosphate pesticides. The OPH6His/UiO-66-NH2 bioconjugate was also tested for the detection of methyl parathion in spiked samples of oranges and tomatoes. The results achieved with our sensing system and their validations with standard HPLC analysis are given in Table 2. The results obtained with the OPH6His/UiO-66-NH2 bioconjugate agree well with standard HPLC results.

3.4.2. Optimization of sensing parameters Several important experimental conditions, such as pH, temperature, and incubation time were optimized to achieve the optimal sensing response from the OPH6His/UiO-66-NH2 system. The pH was varied from 5 to 10, while the temperature effect has been investigated from 20 to 60 °C (Fig. S6). Best sensing response has been obtained at conditions of pH 8 and temperature ∼30–40 °C. Further, the studies on the effect of incubation time (1 mg/mL OPH6His/UiO-66-NH2+ 50 μg/mL methyl parathion, 0–60 min) have proven that the present biosensing system could yield a stable reading within only 5 min (data not shown) which can be termed as a rapid response. The catalytic kinetic of the organophosphorus hydrolase modified

Table 1 A comparison between different OPH enzyme-based optical sensors for the detection of methyl parathion. Nanoplatform

LOD

Dynamic range

Stability

Reference

Graphite-modified carbon electrode CNTs QD/Cys/Au/MWCNT Ni ions modified nitrilotriacetic acid (NTA) agarose F3O4@Au CuInS2 quantum dots BSA/TiO2NFs/AuNPs N-doped carbon dots UiO66-NH2

0.263 μg/mL 0.210 μg/mL 0.997 ng/mL 1 μg/mL 0.1 ng/mL 0.015 μg/mL 7.63 ng/mL 0.09 μg/mL 10 ng/mL

Up to 39 μg/mL 0.5–2.6 μg/mL 5–100 ng/mL 0.26–26 μg/mL 0.5–1000 ng/mL 0.026–10 μg/mL Up to 30.61 μg/mL 0.6–19.14 μg/mL 10–106 ng/mL

45 – 30 – 30 – – – 60

Mulchandani et al. (2001) Deo et al. (2005) Du et al. (2010) Lan et al. (2012) Zhao et al. (2013) Yan et al. (2015) Bao et al. (2017) Song et al. (2017) This work

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days days days

days

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Fig. 6. Results showing (a): reusability of OPH6His/ UiO-66-NH2 bioprobe; (b): long-term storage capacity of OPH6His/UiO-66-NH2 (Formation of PNP was measured by periodically monitoring the efficiency of the stored OPH6His/UiO-66-NH2 for the sensing of methyl parathion (analyte concentration = 50 μg/ mL, bioprobe concentration = 1 mg/mL, T = 37 °C, t = 5 min)); and (c) Specificity of OPH6His/UiO-66NH2 biosensor towards 50 μg/mL methyl parathion in presence of non-specific pesticides (DDT, atrazine and carbamate mix, 100 ppm each).

Science and Engineering Research Board (SERB), Department of Science and Technology (DST, Govt. of India, New Delhi) for financial support (PDF/2016/002182).

Table 2 Application of OPH6His/UiO-66-NH2 for the analysis of spiked amounts of methyl parathion in tomato and orange samples. Sample

Oranges 1 2 3 Tomatoes 1 2 3

Corresponding concentration (μg/mL)

HPLC validation (μg/mL)

Spiked concentration of methyl parathion (μg/mL)

RFI (Relative Fluorescence Index)

1 10 50

0.1496 0.2795 0.4426

1.045 9.89 51.6

1.026 10.17 50.7

1 10 50

0.1458 0.2793 0.4397

0.953 9.86 50.4

1.08 9.89 49.7

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4. Conclusions The present research work demonstrates that OPH6His enzyme can be efficiently conjugated with a functional MOF (i.e, UiO-66-NH2, containing surface eNH2 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 detection of methyl parathion. The pesticide was detectable down to a very low concentration, i.e., 0.19 nM. The used OPH6His/UiO-66-NH2 biomaterial was easily regenerated and then reused for multiple cycles. Its shelf-life was also good. The efficacy of the enzyme-MOF biomaterial demonstrated in the present study should inspire the development of other similar type of MOFs based biocatalytic platforms for environmental sensing and remediation applications. 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 52

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