A magnetic beads-based ligand fishing method for rapid discovery of monoterpene indoles as monoamine oxidase A inhibitors from Hunteria zeylanica.

In this study, a novel magnetic bead-based ligand fishing method was developed for rapid discovery of monoterpene indoles as monoamine oxidase A inhibitors from natural products. In order to improve the screening efficiency, two different magnetic beads, i.e. amine and carboxyl terminated magnetic beads, were comprehensively compared in terms of their ability to immobilize monoamine oxidase A (MAOA), biocatalytic activity and specific adsorption rates for affinity ligands. Carboxyl terminated magnetic beads performed better for MAOA immobilization and demonstrated superior performance in ligand fishing. The MAOA immobilized magnetic beads were applied to screen novel monoamine oxidase inhibitors in an alkaloid-rich plant, Hunteria zeylanica. Twelve MAOA affinity ligands were screened out, and ten of them were identified as monoterpene indole alkaloids by HPLC-Obitrap-MS/MS. Among them, six ligands, namely geissoschizol, vobasinol, yohimbol, dihydrocorynanthenol, eburnamine and (+)-isoeburnamine which exhibited inhibitory activity against MAOA with low IC50 values. To further explore their inhibitory mechanism, enzyme kinetic analysis and molecular docking studies were conducted.

A novel strategy for screening angiotensin-converting enzyme inhibitors from natural products based on enzyme-immobilized ligand fishing combined with active-site blocking and directional enrichment.

Some natural products are important sources of treatments for hypertension based on their potential inhibitory effects on angiotensin-converting enzyme (ACE); however, it is difficult to identify natural ACE inhibitors (ACEIs) due to the complex secondary metabolite environment of natural products. Enzyme immobilization is an important method for screening active constituents in natural products, but this method can sometimes return false-positive and false-negative results. To improve the accuracy and reliability of ligand-fishing methods, we established a novel strategy based on enzyme-immobilized ligand fishing combined with active-site blocking and directional enrichment technologies. We first synthesized ACE-immobilized mesoporous magnetic beads and then verified the screened compounds by molecular docking and in vitro activity detection. We then used active-site blocking to exclude non-specific binding constituents and applied directional enrichment to enrich the low-content constituents for ligand fishing. The screening identified six potential ACEIs from Scutellariae Radix and eight potential ACEIs from Lonicerae japonicae flos, and their inhibitory activity was confirmed by molecular docking simulations and in vitro activity detection. This process screened six additional compounds and excluded two false-positive results as compared with results exclusively using enzyme immobilization. This strategy provides a feasible method for screening active compounds in natural products.

Enzymatic electrochemical biosensor for glyphosate detection based on acid phosphatase inhibition.

A novel enzymatic electrochemical biosensor was fabricated for the indirect detection of glyphosate-based acid phosphatase inhibition. The biosensor was constructed on a screen-printed carbon electrode modified with silver nanoparticles, decorated with electrochemically reduced graphene oxide, and chemically immobilized with acid phosphatase via glutaraldehyde cross-linking. We measured the oxidation current by chronoamperometry. The current arose from the enzymatic reaction of acid phosphatase and the enzyme-substrate disodium phenyl phosphate. The biosensing response is a decrease in signal resulting from inhibition of acid phosphatase in the presence of glyphosate inhibitor. The inhibition of acid phosphatase by glyphosate was investigated as a reversible competitive-type reaction based on the Lineweaver-Burk equation. Computational docking confirmed that glyphosate was the inhibitor bound in the substrate-binding pocket of acid phosphatase and that it was able to inhibit the enzyme efficiently. Additionally, the established method was applied to the selective analysis of glyphosate in actual samples with satisfactory results following a standard method.

Evaluation of Enzyme Inhibitory Activity of Flavonoids by Polydopamine-Modified Hollow Fiber-Immobilized Xanthine Oxidase.

In this study, a polydopamine (PDA)-modified hollow fiber-immobilized xanthine oxidase (XOD) was prepared for screening potential XOD inhibitors from flavonoids. Several parameters for the preparation of PDA-modified hollow fiber-immobilized XOD, including the dopamine concentration, modification time, XOD concentration and immobilization time, were optimized. The results show that the optimal conditions for immobilized XOD activity were a dopamine concentration of 2.0 mg/mL in 10.0 mM Tris-HCl buffer (pH 8.5), a modification time of 3.0 h, an XOD concentration of 1000 µg/mL in 10.0 mM phosphate buffer (pH 7.5) and an immobilization time of 3.0 h. Subsequently, the enzymatic reaction conditions such as the pH value and temperature were investigated, and the enzyme kinetics and inhibition parameters were determined. The results indicate that the optimal pH value (7.5) and temperature (37 °C) of the PDA-modified hollow fiber-immobilized XOD were consistent with the free enzyme. Moreover, the PDA-modified hollow fiber-immobilized XOD could still maintain above 50% of its initial immobilized enzyme activity after seven consecutive cycles. The Michaelis-Menten constant (Km) and the half-maximal inhibitory concentration (IC50) of allopurinol on the immobilized XOD were determined as 0.25 mM and 23.2 µM, respectively. Furthermore, the PDA-modified hollow fiber-immobilized XOD was successfully applied to evaluate the inhibitory activity of eight flavonoids. Quercetin, apigenin, puerarin and epigallocatechin showed a good inhibition effect, and their percentages of inhibition were (79.86 ± 3.50)%, (80.98 ± 0.64)%, (61.15 ± 6.26)% and (54.92 ± 0.41)%, respectively. Finally, molecular docking analysis further verified that these four active compounds could bind to the amino acid residues in the XOD active site. In summary, the PDA-modified hollow fiber-immobilized XOD is an efficient method for the primary screening of XOD inhibitors from natural products.

Effect of Concentrated Salts Solutions on the Stability of Immobilized Enzymes: Influence of Inactivation Conditions and Immobilization Protocol.

This paper aims to investigate the effects of some salts (NaCl, (NH4)2SO4 and Na2SO4) at pH 5.0, 7.0 and 9.0 on the stability of 13 different immobilized enzymes: five lipases, three proteases, two glycosidases, and one laccase, penicillin G acylase and catalase. The enzymes were immobilized to prevent their aggregation. Lipases were immobilized via interfacial activation on octyl agarose or on glutaraldehyde-amino agarose beads, proteases on glyoxyl agarose or glutaraldehyde-amino agarose beads. The use of high concentrations of salts usually has some effects on enzyme stability, but the intensity and nature of these effects depends on the inactivation pH, nature and concentration of the salt, enzyme and immobilization protocol. The same salt can be a stabilizing or a destabilizing agent for a specific enzyme depending on its concentration, inactivation pH and immobilization protocol. Using lipases, (NH4)2SO4 generally permits the highest stabilities (although this is not a universal rule), but using the other enzymes this salt is in many instances a destabilizing agent. At pH 9.0, it is more likely to find a salt destabilizing effect than at pH 7.0. Results confirm the difficulty of foreseeing the effect of high concentrations of salts in a specific immobilized enzyme.

Rapid screening and separation of active compounds against α-amylase from Toona sinensis by ligand fishing and high-speed counter-current chromatography.

In the present study, an efficient method based on ligand fishing and high-speed counter-current chromatography (HSCCC) was established to screen, enrich and separate the active components with the α-amylase inhibitory activity from a traditional dish Toona sinensis. The active components were screened from T. sinensis by ligand fishing using the magnetic immobilized α-amylase prepared through solvothermal and crosslinking methods. HSCCC was used to separate the target compound according to the K value. As a result, a potential active compound 1,2,3,4,6-penta-O-galloyl-ß-d-glucose and a non-target compound quercetin-3-O-α-L-rhamnopyranoside were separated and identified. In-vitro experiments indicated that 1,2,3,4,6-penta-O-galloyl-ß-d-glucose had the activity against α-amylase and the IC50 value was 93.49 ± 0.80 µg/mL which was higher than that of the non-target compound. The result further confirmed the molecular fishing effect of magnetic immobilized α-amylase. The present study can not only find and separate the hypoglycemic substances in T. sinensis quickly and effectively, but also can provide a new approach for the study of natural active components.

Purification, Biochemical Characterization, and Facile Immobilization of Laccase from Sphingobacterium ksn-11 and its Application in Transformation of Diclofenac.

An extracellular laccase enzyme secreted from Sphingobacterium ksn-11 was purified to electrophoretic homogeneity, showing a molecular weight of 90 kDa. The purified enzyme was monomeric in nature confirmed by sodium dodecyl gel electrophoresis. The optimum temperature and pH were found to be 40 °C and 4.5 respectively. The enzyme showed highest substrate specificity for 2,2 azino-bis (ethylthiozoline-6-sulfonate) (ABTS), followed by syringaldazine. The Km value for ABTS was 2.12 mM with a Vmax value of 33.33 U/mg which was higher when compared with syringaldazine and guaiacol substrates. Sodium azide and EDTA inhibited the activity by 30%, whereas presence of Ca2+ and iron increased activity by 50%. The purified enzyme was immobilized in sodium alginate-silicon dioxide-polyvinyl alcohol beads and evaluated for diclofenac transformation studies. LC-MS analysis confirmed that immobilized laccase transformed diclofenac to 4-OH diclofenac after 4 h of incubation. 45 % of diclofenac was able to transform even at 3rd cycle of immobilized laccase use. Therefore, immobilized laccase can be used to transform or degrade several recalcitrant compounds from industrial effluents.

Evaluation inhibitory activity of catechins on trypsin by capillary electrophoresis-based immobilized enzyme microreactor with chromogenic substrate.

In this study, a capillary electrophoresis-based online immobilized enzyme microreactor was developed for evaluating the inhibitory activity of green tea catechins and tea polyphenol extracts on trypsin. The immobilized trypsin activity and other kinetic parameters were evaluated by measuring the peak area of the hydrolyzate of chromogenic substrate S-2765. The results indicated that the activity of the immobilized trypsin remained approximately 90.0% of the initial immobilized enzyme activity after 30 runs. The value of Michaelis-Menten constant (Km ) was (0.47 ± 0.08) mM, and the half-maximal inhibitory concentration (IC50 ) and inhibition constant (Ki ) of benzamidine were measured as 3.34 and 3.00 mM, respectively. Then, the inhibitory activity of four main catechins (epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate) and three tea polyphenol extracts (green tea, white tea, and black tea) on trypsin were investigated. The results showed that four catechins and three tea polyphenol extracts had potential trypsin inhibitory activity. In addition, molecular docking results illustrated that epigallocatechin gallate, epicatechin gallate, epicatechin, and epigallocatechin were all located not only in the catalytic cavity, but also in the substrate-binding pocket of trypsin. These results indicated that the developed method is an effective tool for evaluating inhibitory activity of catechins on trypsin.

Tree bark scrape fungus: A potential source of laccase for application in bioremediation of non-textile dyes.

Although laccase has been recognized as a wonder molecule and green enzyme, the use of low yielding fungal strains, poor production, purification, and low enzyme kinetics have hampered its large-scale application. Thus,this study aims to select high yielding fungal strains and optimize the production, purification, and kinetics of laccase of Aspergillus sp. HB_RZ4. The results obtained indicated that Aspergillus sp. HB_RZ4 produced a significantly large amount of laccase under meso-acidophilic shaking conditions in a medium containing glucose and yeast extract. A 25 µM CuSO4 was observed to enhance the enzyme yield. The enzyme was best purified on a Sephadex G-100 column. The purified enzyme resembled laccase of A. flavus. The kinetics of the purified enzyme revealed high substrate specificity and good velocity of reaction,using ABTS as a substrate. The enzyme was observed to be stable over various pH values and temperatures. The peptide structure of the purified enzyme was found to resemble laccase of A. kawachii IFO 4308. The fungus was observed to decolorize various dyes independent of the requirement of a laccase mediator system.Aspergillus sp. HB_RZ4 was observed to be a potent natural producer of laccase, and it decolorized the dyes even in the absence of a laccase mediator system. Thus, it can be used for bioremediation of effluent that contains non-textile dyes.

Evaluation of xanthine oxidase inhibitory activity of flavonoids by an online capillary electrophoresis-based immobilized enzyme microreactor.

Xanthine oxidase (XOD) is a key enzyme in the human body to produce uric acid, and its inhibitor can be used for the treatment of hyperuricemia and gout. In this study, an online CE-based XOD immobilized enzyme microreactor (IMER) was developed for the enzyme kinetics assays and inhibitor screening. After 30 consecutive runs, the XOD activity remained about 95.6% of the initial immobilized activity. The Michaelis-Menten constant (Km ) of the immobilized XOD was determined as 0.39 mM using xanthine as substrate. The half-maximal inhibitory concentration and inhibition constant of the known inhibitor 4-aminopyrazolo[3,4-d]pyrimidine on XOD were determined as 11.9 and 5.2 µM, respectively. Then, the developed method was applied to evaluate the XOD inhibitory activity of 10 flavonoids, which indicated that dihydroquercetin, quercetin, biochanin A, and epicatechin had significant inhibitory effect on XOD. In addition, molecular docking results verified that the binding energy of the flavonoids with enzyme were in line with their inhibitory activity determined by XOD-IMER. Therefore, the developed XOD-IMER is a potential tool for the primary screening of XOD inhibitors from natural products.

An online dual-enzyme co-immobilized microreactor based on capillary electrophoresis for enzyme kinetics assays and screening of dual-target inhibitors against thrombin and factor Xa.

In this study, an online capillary electrophoresis (CE) based dual-enzyme (thrombin and factor Xa) co-immobilized microreactor (THR-FXa IMER) was constructed for studying enzyme kinetics and screening dual-target inhibitors against THR and FXa with the aid of the polydopamine/graphene oxide (PDA/GO) coating. Based on the developed THR-FXa IMER, the Michaelis-Menten constants (Km) of THR and FXa were calculated to be 187.26 and 48.80 µM, respectively. The inhibition constants (Ki) for two known inhibitors, argatroban and rivaroxaban, on THR and FXa were determined to be 14.73 and 0.41 nM, respectively. In addition, after 30 consecutive runs, the enzymes' activity was remained 98% of the initial immobilized activity for both THR and FXa, which shows that the constructed IMER has good stability and repeatability. Finally, the developed method was successfully applied to screen dual-target inhibitors against THR and FXa from 30 small molecular compounds. Among them, 10 compounds such as salvianolic acid C and epigallocatechin gallate (EGCG) have dual-enzyme inhibitory activity, and 2 compounds named saikosaponin A and oleuropein have single THR inhibitory activity, 5 compounds such as rosemary acid and salvianolic acid B have single FXa inhibitory activity. Finally, the molecular interactions between enzyme and potential inhibitors were further verified via the molecular docking, and a new compound with a theoretically good coagulation inhibition effect was designed by the scaffold hopping study. In summary, the developed THR-FXa IMER is a reliable method for screening THR and/or FXa inhibitors.

"Recent advances on support materials for lipase immobilization and applicability as biocatalysts in inhibitors screening methods"-A review.

With a substantial demand for new anti-obesity drugs for the treatment of obesity, screening lipase inhibitors from natural products has become a popular approach toward drug discovery. Due to the significant advantages of excellent reusability, stability and endurance in extreme pH and temperature conditions, lipase immobilization has been employed as a promising strategy to screen lipase inhibitors. Support is a key factor in the process of enzyme immobilization used to provide excellent biocompatibility, stable physical and chemical properties and abundant binding sites for enzymes. Thus, various supports, including nanofibers, polymeric monoliths, mesoporous materials, nanomaterials, membrane and cellulose paper, are systematically introduced and discussed in this review. Considering these supports, the application of the immobilization of lipase in screening compounds from natural products is also comprehensively reviewed, and the outlook for future research directions is described.

Immobilization of porcine pancreatic lipase onto a metal-organic framework, PPL@MOF: A new platform for efficient ligand discovery from natural herbs.

In terms of ligand fishing, the amount and the relative activity recovery of enzymes immobilized on magnetic particles and nanoparticles are not preeminent. Therefore, the metal-organic framework (MOF) UiO-66-NH2 was synthesized to immobilize the porcine pancreatic lipase (PPL) via precipitation-cross-linking, and the resulting novel biological matrices named PPL@MOF manifested high PPL loading capacity (98.31 mg/g) and relative activity recovery (104.4%). Moreover, the novel enzyme-MOF composite was applied to screen lipase inhibitors from Prunella vulgaris L. to enrich and improve the techniques of ligand fishing. As a result, 13 lipase ligands were obtained, and 12 compounds were determined by HPLC-Q-TOF-MS/MS. All of these ligands were further confirmed to be potential inhibitors through the verification of the activity assay and molecular docking. The proposed approach based on PPL@MOF was superior in terms of abundant protein loading capacity, high enzyme catalytic activity and easy preparation process. Taken together, our newly developed method provided a new platform for efficient discovering bioactive molecules from natural herbs.

Tyrosinase-mediated dopamine polymerization modified magnetic alginate beads for dual-enzymes encapsulation: Preparation, performance and application.

In this study, a simple and efficient method to obtain entrapment of mixtures of double enzymes is developed. As a proof of principle, double enzymes (tyrosinase (TYR) and ß-glucosidase (ß-Glu)) were co-immobilized in magnetic alginate-polydopamine (PDA) beads using in situ TYR-mediated dopamine polymerization and internal setting strategy-mediated magnetic alginate-PDA gelation. The leakage of enzymes from the magnetic alginate beads was significantly reduced by exploiting the double network cross-linking of alginate and PDA, which was induced by the d-(+)-Gluconic acid δ-lactone (GDL) and TYR, respectively. The physicochemical properties of the prepared magnetic alginate beads were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) analysis. After that, the enzymatic reaction conditions and the performance of the entrapped TYR and ß-Glu, such as enzyme kinetics and inhibition kinetics, were investigated. The Michaelis-Menten constants (Km) of the entrapped TYR and ß-Glu were determined as 2.72 and 3.45 mM, respectively. The half-maximal inhibitory concentrations (IC50) of kojic acid and castanospermine for the entrapped TYR and ß-Glu were determined as 13.04 and 56.23 µM, respectively. Finally, the entrapped double enzymes magnetic alginate beads were successfully applied to evaluate the inhibitory potency of six kinds of tea polyphenols extracts. Black tea and white tea showed high inhibition activity against TYR were (36.14 ± 1.43)% and (36.76 ± 2.35)%, respectively, while the black tea and dark tea showed high inhibition activity against ß-Glu were (37.89 ± 6.70)% and (21.28 ± 4.68)%, respectively.

Magnetic nanoparticles-based lactate dehydrogenase microreactor as a drug discovery tool for rapid screening inhibitors from natural products.

Lactate dehydrogenase (LDH), catalyzing the conversion of pyruvate to lactate during glycolysis, is overexpressed in cancer cells. LDH inhibitors are a promising approach for the treatment of cancer. But up till now, there is limited method for rapid screening of LDH inhibitors. Herein, the use of LDH functionalized magnetic nanoparticles as a drug discovery tool for the selective enrichment of LDH potential inhibitors from natural products was firstly reported in this study. Firstly, LDH was immobilized onto the surface of amino-modified magnetic nanoparticles via covalent binding. In order to obtain the maximum enzyme activity, the immobilization conditions including pH, time and LDH concentration were optimized. The amount of LDH immobilized on MNPs was about 49 µg enzyme/mg carrier under the optimized conditions. Subsequently, the ligand fishing assay was performed to validate the specificity and selectivity of immobilized LDH using a model mixture, which consisted of galloflavin, chlorogenic acid and verbascoside. Finally, the immobilized LDH approach combined with ultra-high performance liquid chromatography-tandem mass spectrometry technique (UHPLC-MS/MS) was applied to screen potential LDH inhibitors from two anthraquinone-rich natural products (Rhubarb and Polygonum cuspidatum). Nine and six compounds were identified from Rhubarb and Polygonum cuspidatum extracts respectively, of which three compounds were common to both. Our results have proven that LDH functionalized magnetic nanoparticles have a significant prospect for drug discovery from complex matrices.

Urease immobilization on magnetic micro/nano-cellulose dialdehydes: Urease inhibitory of Biginelli product in Hantzsch reaction by urea.

Micro/nano celluloses (MC)/NC) were magnetized by nanoγ-Fe2O3 into the nanoγ-Fe2O3@MC (NMMC) and nanoγ-Fe2O3@NC (NMMC) which oxidized to NMMCD and NMNCD dialdehydes for Schiff-base immobilization of urease as NMMCD/urease and NMMCD/urease. The relative enzyme-activity of the immobilized ureases were comparable with the free-urease, although 75%-80% of the enzyme activity preserved for NMMCD/urease and NMNCD/urease after six cycles. The compared catalytic activities of the NMMCD/urease and NMMCD/urease in Biginelli/Hantzsch reactions in water at 60 °C surprised us by 100% selectivity for the Biginelli product 3,4-dihydropyrimidin-2(1H)-one (DHPM1). With the superiority of NMNCD/urease, this high selectivity using immobilized ureases is owing to the admirable urease inhibitory of the formed Biginelli product DHPM1 by urea condensation instead of urea hydrolysis. The robust enzyme inhibitory of the DHPM1 for free urease was also confirmed by phenol red test to show the deactivation of enzyme for enzymatic hydrolysis of urea and ammonia production in water.

Rapid on-line system for preliminary screening of lipase inhibitors from natural products by integrating capillary electrophoresis with immobilized enzyme microreactor.

An on-line system for preliminary screening lipase inhibitors from natural products with an immobilized lipase microreactor coupled to capillary electrophoresis was established. In this research, the lipase was anchored on the amino activated capillary inner wall using glutaraldehyde as a homobifunctional linker through Schiff base reaction. The immobilized lipase activity was evaluated by measuring the peak area of the hydrolyzate of p-nitrophenyl acetate. In order to maintain the enzymatic activity of immobilized lipase, the acetonitrile content and the pH of the reaction solution were also optimized. Under the optimized reaction conditions, the Michaelis-Menten constant of the immobilized lipase and the half maximal inhibitory concentration for orlistat were studied, which were consistent with previous literature data. Furthermore, the developed method was applied to screen lipase inhibition activity from ten natural products. As a result, we found that six natural products have inhibitory effect on the activity of lipase, among which the inhibitory effect of Rhizoma atractylodis extract has never been reported before.

Identification of ligands from natural products as inhibitors of glutathione S-transferases using enzyme immobilized mesoporous magnetic beads with high-performance liquid chromatography plus quadrupole time-of-flight mass spectrometry and molecular docking.

Multidrug resistance is recognized as one of the main reasons leading to the failure of chemotherapy. Studies have shown that glutathione S-transferase inhibitors could be regarded as multidrug resistance reversal agents. Herein, a method of applying enzyme immobilization, molecular docking, and high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was employed to screen glutathione S-transferase inhibitors from natural products. Magnetic mesoporous silica microspheres were synthesized and modified with a poly(dopamine) layer, which has a large quantity of amino, enabling further non-covalent binding with glutathione S-transferase. Moreover, the immobilization conditions, namely, potential of hydrogen, catalase concentration, reaction temperature and reaction time, were optimized. In total, six potential compounds were isolated and identified from Perilla frutescens (L.) Britt leaves and green tea and molecular docking was applied to identify the binding site. Rosmarinic acid, (-)-epigallocatechin-3-O-gallate and (-)-epicatechin-3-gallate showed higher binding affinity than the compounds, and their half maximal inhibitory concentration values were further determined. The results suggested that this proposed method was effective and convenient for identifying glutathione S-transferase inhibitors from natural products.

An l-glutaminase enzyme reactor based on porous bamboo sticks and its application in enzyme inhibitors screening.

Inspired by the porous and fibrous structure of commercially available bamboo, herein we created an l-glutaminase enzyme reactor based on bamboo sticks. The enzyme was immobilized onto the bamboo sticks through a glutaraldehyde modification to achieve covalent bonding. The enzymatic hydrolysis efficiency of the prepared l-glutaminase@bamboo sticks based porous enzyme reactor was evaluated by chiral ligand exchange capillary electrochromatography using l-glutamine as the substrate. l-glutaminase@bamboo exhibited improved enzymatic hydrolysis performances, including high hydrolysis efficiency (maximum rate Vmax: two fold higher than the free enzyme), prolonged stability (14 days) and good reusability. l-Glutaminase@bamboo sticks also expanded application capability in pharmaceutical industry in enzyme inhibitor screening. These excellent properties could be attributed to the micropores of bamboo sticks, which led to the fast enzymatic kinetics. The results suggest that the pores of bamboo sticks played an important role in the proposed enzyme reactor during the hydrolysis of l-glutamine and l-glutaminase inhibitor screening.

Amperometric biosensor for glyphosate based on the inhibition of tyrosinase conjugated to carbon nano-onions in a chitosan matrix on a screen-printed electrode.

Glyphosate [N-(phosphonomethyl)glycine] is the most frequently used herbicide to date. Due to its indiscriminate use, it has become a globally occurring pollutant of surface waters. A biosensor for glyphosate is described here that consists of a carbon nano-onion/tyrosinase conjugate immobilized in a chitosan matrix on a screen-printed electrode. The analytical principle is based on the inhibition of the enzyme tyrosinase by glyphosate. L-DOPA is used as the enzyme substrate. The presence of the carbon nano-onions has a beneficial effect on the sensitivity of the assay. Glyphosate can be amperometrically quantified in the 0.015 to 10 µM concentration range and with a 6.5 nM (1.1 µg L-1) detection limit. The biosensor is stable more than 2 months at 4 °C. It was applied to the detection of glyphosate in water and soil samples taken from irrigation of a rice field after aerial application. Results were in good agreement with data obtained by a commercial ELISA. Graphical abstract A highly sensitive amperometric biosensor for glyphosate is reported, based on the covalent immobilization of a carbon nano-onion/tyrosinase conjugate on a chitosan matrix.