Design of enzyme-immobilized polymer brush-grafted magnetic nanoparticles for efficient nematicidal activity

Abstract

Parasitic nematodes not only cause deadly diseases in plants and animals but also adversely affect agricultural industry and global health, particularly in developing countries. In this study, we planned to combine the concept of enzyme immobilization with nanotechnology to develop magnetic nanoparticles (MNPs) with efficient nematicidal activity in water. A novel nematicidal platform was developed by immobilizing protease (from Streptomyces griseus) on the surface of polymer brush-grafted magnetic nanoparticles (MNPs–PGMA–Pro). For comparison, a monolayer-based nematicidal platform was also developed by functionalizing protease on the surface of glutaraldehyde-functionalized MNPs (MNPs–GA–Pro). MNPs–PGMA–Pro particles show enhanced enzyme activity and stability over a wide range of temperature and pH, as compared to MNPs–GA–Pro. Polymer brush- and monolayer-based protease-functionalized MNPs exhibit superior enzyme activity when compared to the free enzyme. When tested for nematicidal activity against parasitic nematodes (Haemonchus contortus), the polymer brush-based platform retained higher activity over 7 cycles of magnetic separation. The reported platforms can be prospectively employed for water treatment, whereas their reusability over many remediation cycles due to facile magnetic separation promises a substantially reduced treatment cost.

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1. Introduction

Recently, nanotechnology has attracted substantial scientific interest due to its applications in almost all the facets of everyday life. Among nanoparticles, Fe₃O₄ magnetic nanoparticles (MNPs) are widely employed as carriers of bioactive molecules such as enzymes and drugs, and they exhibit interesting attributes of large surface area, biocompatibility, convenient magnetic separation and drug delivery.^1,2 Various methods have been developed for the immobilization of enzymes on the surface of MNPs such as adsorption or covalent conjugation to carboxylic acid, aldehyde, thiol, epoxide, and maleimide groups.^3,4 Polymer brushes, which are defined as surface-tethered polymeric chains, are excellent candidates for modulating the surface chemical and physical properties of a wide variety of materials. The key advantages associated with polymer brushes when compared to other surface functionalization strategies include higher stability and activity, which are inherent in their covalent nature and the higher density of active groups on the surface.

Bio-inspired materials and bioactive natural products have been widely explored for biocatalytic applications due to their inherent activity and biocompatibility. Protease enzymes are well recognized for their proteolytic activities and are employed for proteomic analysis, hydrolysis of proteins in a variety of substrates such as wool and rapeseed, and enzymatic degradation of synthetic polymers.^3,5–7 Proteases are reported to be secreted by some fungal and bacterial species as a biological control against nematodes, where nematicidal activity is achieved by destroying the cuticle of the target.⁸ Thus, naturally isolated protease enzymes are attractive candidates as nematicides. The stability and activity of enzymes are fundamentally affected by various physiological and environmental parameters. Immobilization of an enzyme onto a surface is an affecting strategy for enhancing the stability, activity and, in some cases, reusability of the enzyme.^9–11

Parasitic nematodes are not only known for their harmful impact on plants and animals but are also recognized for causing many deadly diseases in humans.¹² Their free-living species are abundant in fresh and saltwater bodies and soil habitats. A higher concentration of nematodes in drinking water adds an unpleasant taste to the drinking water and these nematodes may carry pathogenic bacteria in their gut.¹³ In this study, we tested Haemonchus contortus as a sample nematode in drinking water because it is a blood-feeding nematode that affects ruminants and is responsible for anaemia and death of grazing animals, and thus causes a serious impact on the livestock industry.^14,15

Considering these problems, in the present study, extracellular protease enzyme was covalently immobilized on the surface of polymer brush-functionalized MNPs. Poly(glycidyl methacrylate) (PGMA) brushes were grafted onto the surface of MNPs particles by surface-initiated atom transfer radical polymerization (SI-ATRP), followed by covalent binding of the enzyme. For comparison, a monolayer-based platform was also developed by immobilizing protease onto the surface of glutaraldehyde-modified MNPs. The polymer brush-based platform exhibited superior nematicidal activity and stability of the enzyme over a wide range of pH and temperature as compared to the monolayer-based platform and free protease.

2. Experimental section

2.1. Materials

Peptone (98%), yeast extract (99%), NaCl (98%), casein (99.9%), glucose (98%), soybean meal (99%), KH₂PO₄ (99%), MgSO₄ (99%), Na₂CO₃ (99%), trichloroacetic acid (TCA) (99%), alkaline reagent (99%), Folin's reagent (99%), FeSO₄ (>99%), KNO₃ (99%), KOH (>90%), ethanol (>99%), NaH₂PO₄ (97%), Na₂HPO₄ (98%), ammonia solution (35%), (3-aminopropyl) triethoxysilane (APTES) (99%), glutaraldehyde (50% in water), tetraethyl orthosilicate (TEOS) (99%), α-bromoisobutyryl bromide (99%), glycidyl methacrylate (GMA) (99%), bipyridyl, Cu(II)Br₂ (99%), and Cu(I)Br (99%) were purchased from Sigma Aldrich, Germany. All the reagents were used without further purification. Protease (PRONASE® protease, Streptomyces griseus) was purchased from Merck KGaA, Germany.

2.2. Immobilization of protease on magnetic nanoparticles (MNPs–GA–Pro)

FeSO₄·7H₂O (20 g, 72 mmol) was dissolved in 140 mL deionized water, pre-degassed with nitrogen, and the temperature was adjusted to 90 °C. A solution of KNO₃ (1.62 g, 16 mmol) and KOH (11.23 g, 200 mmol) was prepared in 60 mL H₂O. This solution was added dropwise to the above mentioned solution in approximately 5 min under nitrogen bubbling. The reaction mixture was heated to 90 °C with mechanical stirring for 1 h. The solution was allowed to stand overnight at room temperature. MNPs were separated magnetically and washed several times with deionized water. For coating with silica, MNPs (200 mg) were dispersed in 150 mL ethanol, and the solution was sonicated for 15 min in ice. Ammonia solution (12 mL, 35%) and TEOS (400 μL, 1.8 mmol) were added to the suspension, and the mixture was sonicated in an ice bath for 2 h. The particles (MNPs–OH) were separated from the reaction mixture using a permanent magnet and washed thoroughly with ethanol. For the preparation of amine-functionalized nanoparticles, APTES (13 mL, 55.5 mmol) was added in ethanol (320 mL) followed by addition of MNPs–OH particles (1 g). The mixture was mechanically stirred for 3 h at 40 °C. Black particles (MNPs–NH₂) were separated from the reaction mixture and washed thoroughly with ethanol.

MNPs–NH₂ particles (200 mg) were dispersed in 5% solution of glutaraldehyde (GA) in 0.1 M phosphate buffer solution (25 mL, pH = 7). The mixture was stirred at room temperature for 6 h. The nanoparticles (MNPs–GA) were washed thoroughly with phosphate buffer and separated by a magnet.

Immobilization of proteases on MNPs–GA particles was carried out using protease enzyme (20 mg) in phosphate buffer solution (20 mL, pH = 7). MNPs–GA particles (110 mg) were dispersed in the above mentioned solution and placed on a shaker for 12 h at room temperature. Treated particles were separated from the reaction mixture using a permanent magnet and washed with deionized water.

2.3. Immobilization of enzyme on polymer brush-grafted magnetic nanoparticles (MNPs–PGMA–Pro)

Briefly, a solution of TEA (120 μL, 0.86 mmol) and α-bromoisobutyryl bromide (90 μL, 0.72 mmol) in DCM (7 mL) was degassed for 15 min at room temperature and injected over degassed MNPs–NH₂ (330 mg) particles under an inert atmosphere. The reaction mixture was mechanically stirred under an inert atmosphere for 2.5 h at room temperature. The particles (MNPs–BI) were separated by a permanent magnet and washed twice with ethanol and DCM.

GMA (5.7 mL, 42.9 mmol) was dissolved in DMF : H₂O (12 mL, 2 : 1), followed by the addition of bipyridyl (83 mg, 0.532 mmol) and Cu(II)Br₂ (11.7 mg, 0.052 mmol). After degassing for 1 h, Cu(I)Br (40 mg, 0.28 mmol) was added and the solution was degassed for another 15 min. The above mentioned solution was injected over MNPs–BI under an inert environment. Polymerization was carried out for 12 h at room temperature and subsequently the particles (MNPs–PGMA) were washed 3 times with acetone and THF.

Proteases were immobilized on polymer brush-grafted fabricated MNPs by dispersing MNPs–PGMA (110 mg) in a PBS (20 mL, pH = 7) solution of protease enzyme (20 mg). The suspension was shaken for 12 h at room temperature and particles were separated using a permanent magnet followed by repeated washings with deionized water.

2.4. Characterization

Scanning electron microscopy (SEM) was performed on a Hitachi SU8000 SEM. Transmission electron microscopy (TEM) was carried out on Philips EM 420 and FEI Tecnai G2 F30 instruments. A physical property measurement system (PPMS) (Cryogenic, VSM 9 T Magnet) was used at 295 K to measure magnetic properties of MNPs. ATR-FTIR spectra were recorded on an ATR-FTIR Alpha Bruker spectrometer. Thermogravimetric analysis (TGA) was performed with a TA Instruments device (Model TGA Q500) from room temperature to 1000 °C at a heating rate of 10 °C min⁻¹ under a nitrogen gas (N₂) atmosphere (40 mL min⁻¹). Other instruments include an incubator (EHRET BK4444, Germany), a spectrophotometer (Thermo Nicolet 300, USA), and a centrifuge (Sigma 2-6, USA). The zeta potentials and hydrodynamic radii of the MNPs in phosphate buffered saline (PBS) were measured with a Malvern ZetaNano ZS at 25 °C. A U-shaped DTS1070 capillary cell was used to estimate the zeta potential using a He–Ne laser source of 5 mW at 633 nm. Each sample was measured three times, and the values reported (Table SI-2†) are an average of these measurements.

2.5. Enzyme essay

Free and immobilized protease were assayed by the method proposed by McDonald and Chen.¹⁶ Casein (4 mL) was added to a suitable amount of enzyme and incubated at 37 °C for 30 min. 1% trichloroacetic acid (1 mL) was added to stop the reaction. The reaction mixture was centrifuged at 6000 rpm for 10 min. Alkaline reagent (5 mL), 1 N aq. NaOH (1 mL) and Folin's reagent (1 mL) were added to the supernatant (1 mL). The absorbance of the blue-colored complex was measured with a UV/vis spectrophotometer at 700 nm. One unit of protease activity (U) is defined as the amount of enzyme that is required to cause an increase of 0.1 in optical density at 700 nm under defined conditions. The effects of physical parameters on enzyme activity were determined by taking samples from the reaction mixture after 0, 12, 24, 36 and 48 h under different experimental conditions (temperature and pH).

2.6. Effect of temperature and pH on protease activity

The effect of temperature on the activity of free and immobilized proteases was studied. Both free and immobilized proteases were incubated at different temperatures ranging from 25 to 80 °C. The amount of free enzyme used was 1 mg in each case, whereas the amount of MNPs–PGMA–Pro employed was 5.5 mg. 5.5 mg amount of MNPs–PGMA–Pro was chosen because at this amount the nematicidal activity of MNPs–PGMA–Pro was comparable to the nematicidal activity of 1 mg free enzyme. The activity was measured by performing a standard assay method. Similarly, the effect of pH of the medium on the activity of the enzyme was investigated by incubating free (1 mg) and immobilized protease (5.5 mg) in PBS solutions of different pH values ranging from 5 to 12. The activity of the enzyme was measured by incubating casein and protease in PBS solutions of different pH at 37 °C. The activity was measured by performing a standard assay method.

2.7. Nematicidal activity of immobilized proteases (MNPs–GA–Pro and MNPs–PGMA–Pro)

The bioactivity of free and immobilized proteases was determined using a standard assay method at regular intervals to check the effect of particles on nematodes. Nematodes were washed thoroughly with PBS (pH = 7.0) before use. 30 μL of nematode stock solution (containing approximately 50–60 nematodes) was transferred into a 1.5 mL Eppendorf tube. Functionalized MNPs (MNPs–GA–Pro and MNPs–PGMA–Pro) (1 mg) were added and the mixture was incubated at 37 °C for 12, 24, 36 and 48 h. As a control, blank samples were run parallel to immobilized proteases under the same conditions. The effect of immobilization on the efficiency of proteases was investigated by observing the numbers of living and dead nematodes under a light microscope, which was later confirmed using a scanning electron microscope.

3. Results and discussion

MNPs particles with an average size of ∼100 nm were prepared by simple alkaline hydrolysis of FeSO₄. MNPs were suspended in ethanol and treated with TEOS under basic conditions. This treatment resulted in silica-coated MNPs. Coating of silica on MNPs was performed to facilitate the subsequent silanization process employing APTES, which resulted in amine-functionalized MNPs (MNPs–NH₂).¹⁷ The surface amino groups of MNPs–NH₂ were reacted with glutaraldehyde (GA) under ambient conditions, resulting in MNPs with surface aldehyde groups (MNPs–GA).¹⁸ The surface aldehyde groups of MNPs–GA were conjugated to the amino groups of protease via imine linkages, resulting in a monolayer-based protease-functionalized MNPs platform (MNPs–GA–Pro). For the fabrication of protease-grafted polymer brushes-based MNPs platform, PGMA brushes were grown from the surface of ATRP initiator-functionalized MNPs (MNPs–BI) by SI-ATRP in the presence of a Cu(I)/Cu(II)–BiPy catalyst system.¹⁹ The complete functionalization strategy for MNPs is illustrated in Scheme 1.

Scheme 1 Schematic illustration of protease immobilization onto the surface of monolayer and polymer brush-functionalized magnetic nanoparticles.

All the surface functionalization steps were monitored by ATR-FTIR spectroscopy. MNPs show a typical absorption band at 546 cm⁻¹, which corresponds to Fe–O stretching vibrations. The coating of silica on MNPs after the treatment with TEOS was confirmed by the characteristic absorption band for Si–O–Si stretching vibrations at 1049 cm⁻¹.¹⁷ In the case of a monolayer-based platform, the immobilization of protease on MNPs–GA was supported by the absorption band at 1647 cm⁻¹ due to the stretching vibrations of carbonyl groups in protease. For the polymer brush-based platform, the successful growth of polymer brushes for MNPs–PGMA was confirmed by the absorption band at 1731 cm⁻¹, which originates from the stretching vibrations of carbonyl (C=O) groups in PGMA. The subsequent conjugation of protease via a reaction with the epoxide groups of PGMA was supported by the absorption bands at 1631 cm⁻¹ and 3300 cm⁻¹, which are due to amide and N–H bond stretching, respectively (Fig. 1).¹⁹

Fig. 1 ATR-FTIR spectra of MNPs–NH₂ (a), MNPs–GA–Pro (b), MNPs–PGMA (c), and MNPs–PGMA–Pro (d).

The successful chemical modification of the surface of MNPs was further corroborated by XPS analysis (Fig. 2). The magnetite (Fe₃O₄) nature of the MNPs used in this study was confirmed by the binding energy signals of Fe 2p_1/2 and Fe 2p_3/2 at 725 eV and 711 eV (Fig. SI-1a†), respectively. The functionalization of MNPs by APTES resulted in the appearance of characteristic signals for silicon at 153 eV and 103 eV for Si 2s and Si 2p, respectively. In addition, the peak at 400 eV for N 1s substantiated the successful functionalization of MNPs surfaces with APTES (Fig. SI-1b†).¹⁷ The conjugation of surface amino groups in MNPs–NH₂ with glutaraldehyde brings about a growth in carbon content at the surface of MNPs, which was substantiated by a decrease in the N/C ratio from 0.079 in MNPs–NH₂ to 0.046 in MNPs–GA. An increase in the carbon content (C 1s) during enzyme immobilization confirmed the successful fabrication of monolayer-based protease-functionalized MNPs (Fig. 2c). The grafting of an ATRP initiator, which is required for SI-ATRP of PGMA, on the surface of MNPs–NH2 was validated by the appearance of signals for bromine at 255 eV, 182 eV and 70 eV for Br 3s, Br 3p and Br 3d, respectively. The fabrication of PGMA brushes on the surface of MNPs was verified by the disappearance of Br signals and a substantial decrease in the N 1s signal intensity, as PGMA does not contain any nitrogen; this further led to a decrease in the N/C ratio from 0.078 in MNPs–Br to 0.02 in MNPs–PGMA (Fig. 2d). Immobilization of protease on MNPs–PGMA surface was confirmed by an increase in surface nitrogen content, as revealed by the higher intensity of the N 1s signal with a concomitant increase in the N/C ratio to 0.082 (Fig. 2e).

Fig. 2 XPS survey scans of MNPs–BI (a), MNPs–GA (b), MNPs–GA–Pro (c), MNPs–PGMA (d), and MNPs–PGMA–Pro (e) and magnetization curves for MNPs, MNPs–GA and MNPs–PGMA (f).

The magnetization of MNPs under an applied magnetic field is known to decrease with increase in the thickness of the surface coating because of the shielding effect. Unmodified MNPs exhibited the highest magnetization, which decreased in the case of monolayer-functionalized MNPs. Polymer brushes-functionalized MNPs displayed the lowest magnetization, which can be related to an increased shielding effect that is inherent in the thick polymer brush layer. The traces of hysteresis loop for the magnetization of MNPs under an applied magnetic field fully corroborated the surface functionalization of MNPs (Fig. 2f).

SEM and TEM images of MNPs revealed particles with a size of ∼100 nm with sharp and clean edges (Fig. 3a). A thin layer of ∼5 nm thickness was observed to cover the surface of monolayer-functionalized particles (MNPs–GA), whereas functionalization with PGMA polymer brushes led to a uniform coating of a thicker layer of ∼20 nm thickness on the surface of nanoparticles. The deposition of larger amounts of material on the surface of polymer brushes-functionalized nanoparticles is also confirmed by thermogravimetric analysis (TGA, Fig. SI-2†). In TGA, pristine MNPs did not exhibit any significant change in mass. The GA-functionalized MNPs lose about 1.17% of their mass when heated to 900 °C. MNPs–GA–Pro exhibits slightly higher (2.30%) weight loss, which can be attributed to the surface-immobilized protease. On the other hand, a considerably larger amount of organic content was evident from the TGA analysis of polymer brush-coated MNPs. MNPs–PGMA exhibited significantly higher weight loss (66.72%), whereas MNPs–PGMA–Pro exhibited a total weight loss of 70.3%. From the difference in weight loss, it can be inferred that the enzyme content of MNPs–PGMA–Pro is 3.2 times higher than the enzyme content of MNPs–PGMA.

Fig. 3 SEM image of MNPs (a), HRTEM image of MNPs (b) and TEM images of MNPs–GA (c) and MNPs–PGMA (d).

MNPs–GA showed a zeta potential of −11.67 ± 1.13 mV. A slightly higher zeta potential (−14.11 ± 1.02 mV) was observed for MNPs–GA–Pro, which could be due to the larger negative charge imparted to the surface by the immobilized enzyme. In the case of MNPs–PGMA, the immobilization of protease on the polymer brush layer enhanced the particle dispersion more drastically and increased the zeta potential from −10.24 ± 0.75 to −17.20 ± 1.05 mV. Immobilization of enzymes on MNPs significantly reduced the hydrodynamic size of the MNPs, reflecting an increase in surface hydrophilicity and better dispersion in an aqueous medium. The increase in zeta potential and decrease in the hydrodynamic size of MNPs can be related to the enzyme diffusion layer on the surface of MNPs. Before immobilization of enzymes, monolayer GA and PGMA brush-coated MNPs form large aggregates, as observed in SEM images (Fig. 3a). However, the immobilization of enzymes on the surface possibly decreases the agglomeration of MNPs, leading to a smaller hydrodynamic diameter and higher zeta potentials.

The stability and activity of enzymes largely depend on temperature. Therefore, the effect of temperature on the activity of free and immobilized protease was studied in the temperatures range from 25 °C to 80 °C (Fig. SI-3†). The maximum activity of free (43.14 U mL⁻¹) and immobilized enzyme (MNPs–GA–Pro, 45.27 U mL⁻¹ and MNPs–PGMA–Pro, 47.6 U mL⁻¹) was observed at 37 °C. Above 40 °C, the activity of both free and immobilized proteases started to decline.

At 60 °C, immobilized enzymes (MNPs–GA–Pro and MNPs–PGMA–Pro) retained more than 65% of their activity, whereas the free enzyme retained only 26% of its activity. At 80 °C, both MNPs–GA–Pro (23%) and MNPs–PGMA–Pro (35%) exhibited noticeable catalytic activity, whereas the free protease was completely inactive at this temperature. This suggests that the polymer brush-based platform (MNPs–PGMA–Pro) was more effective in retaining enzymatic activity over the temperature range of 25 °C–80 °C.

The pH of the medium has a significant impact on the activity of enzymes. The activity of free and immobilized protease (MNPs–GA–Pro and MNPs–PGMA–Pro) was assessed over the pH range of 5–12 at 37 °C (Fig. SI-4†). The results showed that the activity of free protease was more adversely affected by a change in pH as compared to immobilized enzyme. The maximum activity of free and immobilized proteases was found at pH = 7.0. Above and below pH = 7.0, the activity of enzymes started to decline. At pH 5.0, the activity of free protease was only 25%, whereas immobilized protease retained 51% (MNPs–GA–Pro) and 63% (MNPs–PGMA–Pro) of their activity. As observed for the effect of temperature variation, the polymer brush-based platform (MNPs–PGMA–Pro) demonstrated better performance over a wide range of pH as compared to the free enzyme.

The bioactivity of free and immobilized proteases was investigated against the nematode species Haemonchus contortus. Nematicidal activity revealed that about 80%, 84% and 94% of nematodes were dead after 48 h with free protease, MNPs–GA–Pro and MNPs–PGMA–Pro, respectively. In the case of the control experiment (without enzyme), bare MNPs and particles with intermediate functionalizations (MNPs–NH₂, MNPs–GA, and MNPs–PGMA) did not show any significant activity and >80% of nematodes were alive with an intact cuticle even after 48 h (Table SI-1†).

On treatment with MNPs–GA–Pro, nematodes were dead after 12 h, their cuticle started shrinking followed by appearance of holes on the cuticle within 24 h, and after 48 h, their body was divided into fragments (Fig. 4).

Fig. 4 Bioactivity of MNPs–GA–Pro particles against Haemonchus contortus. (a) Live nematode in control sample. (b) Nematodes were dead after 12 h and cuticle started shrinking. (c) After treatment for 24 h, holes appeared on cuticles. (d) After 48 h, body was divided into fragments.

In the case of MNPs–PGMA–Pro particles, nematodes were dead after 12 h and cuticles started to rupture after 24 h, followed by the complete destruction of cuticles in 36 h (Fig. 5). Therefore, polymer brush-grafted particles not only manifested enhanced stability but also demonstrated significantly higher nematicidal activity.

Fig. 5 Bioactivity of MNPs–PGMA–Pro particles against Haemonchus contortus. (a) Healthy nematodes in control group. (b) Nematodes were dead and curves developed after 12 h. (c) Cuticle started rupturing after 24 h. (d) Cuticle was completely ruptured after 36 h.

The advantage of immobilizing protease enzymes on MNPs surface is attributed to their reusability via facile magnetic separation. The reusability of immobilized protease was examined up to 7 catalytic cycles (Fig. 6). After 3 cycles, the activity of MNPs–GA–Pro started to decline rapidly, whereas polymer brush-functionalized MNPs–PGMA–Pro particles retained significant activity even after 7 cycles of magnetic separation. This gradual decrease in activity may be attributed to the reversibility of imine bond formation, which may lead to the dissociation of surface-immobilized enzymes over time and/or the denaturation of enzymes.²⁰ Loss of particles during the separation process and desorption of any physisorbed enzyme may also contribute towards the decrease in the activity.

Fig. 6 Reusability of immobilized protease against Haemonchus contortus.

4. Conclusions

In the present study, nematicidal activity of protease immobilized on the surface of MNPs is reported. To compare the effects of surface functionalization methodologies on the enzymatic activity, the surface immobilization of enzymes was carried out via monolayer and polymer brush routes. Compared to free protease, the protease that was covalently immobilized on the surface of MNPs (MNPs–PGMA–Pro and MNPs–GA–Pro) displayed higher nematicidal activity and exhibited greater stability over a wide pH and temperature range. The facile magnetic separation of enzyme-functionalized MNPs from their suspension in water facilitated reusability of the enzyme. Because of the higher density of surface functional groups, proteases immobilized on the surface of polymer brush-functionalized MNPs exhibited superior enzymatic activity over 7 cycles of magnetic separation when compared to a monolayer-based platform. The present study provides an effective strategy for the future development of magnetically separable and reusable antimicrobial platforms that may be potentially employed for water remediation.

Acknowledgements

B.Y. acknowledges funding from HEC, Pakistan (Project No. 20-1740/R&D/10/3368 and 20-1799/R&D/10-5302) and LUMS start-up grant. H.D. gratefully acknowledges financial support from TUBITAK (Project No. 112M804).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: TGA details, XPS data (of bare MNPs and MNPs–NH₂), and mortality rate of nematodes are provided in the ESI. See DOI: 10.1039/c5ra10063a

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