ABSTRACT
Redox nanozymes have exhibited various applications in recognizing environmental pollutants but not aromatic amines (a type of typical pollutant). Herein, with Cu2+ as a node and tryptophan (Trp) as a linker, Cu-Trp as a specific ascorbic acid oxidase mimic was synthesized, which could catalyze ascorbic acid (AA) oxidation to dehydroascorbic acid (DHAA). Alternatively, with other natural amino acids as linkers to synthesize Cu-based nanozymes, such catalytic performances are also observed. The as-produced DHAA could react with o-phenylenediamine (OPD) and its derivatives (2,3-naphthalene diamine (NDA), 4-nitro-o-phenylenediamine (4-NO2-OPD), 4-fluoro-o-phenylenediamine (4-F-OPD), 4-chloro-o-phenylenediamine(4-Cl-OPD), and 4-bromo-o-phenylenediamine(4-Br-OPD)) to form a Schiff base and emit fluorescence. Based on the results, with Cu-Trp + AA and Cu-Arg (with arginine (Arg) as a linker) + AA as two sensing channels and extracted red, green, and blue (RGB) values from emitted fluorescence as read-out signals, a visual sensor array was constructed to efficiently distinguish OPD, NDA, 4-NO2-OPD, 4-F-OPD, 4-Cl-OPD, and 4-Br-OPD as low as 10 µM. Such detecting performance was further confirmed through discriminating binary, ternary, quinary, and senary mixtures with various concentration ratios, recognizing 18 unknown samples, and even quantitatively analyzing single aromatic amine. Finally, the discriminating ability was further validated in environmental waters, providing an efficient assay for large-scale scanning levels of multiple aromatic amines.
Subject(s)
Amines , Ascorbate Oxidase , Schiff Bases , Schiff Bases/chemistry , Amines/chemistry , Amines/analysis , Ascorbate Oxidase/chemistry , Ascorbate Oxidase/metabolism , Copper/chemistry , Copper/analysis , Ascorbic Acid/chemistry , Ascorbic Acid/analysis , Phenylenediamines/chemistry , Oxidation-ReductionABSTRACT
Copper metalloenzymes ascorbate oxidase (AOase), amine oxidase (AmOase), and catechol oxidase (COase) possess copper(II) sites of coordination, which are trimeric, homodimeric, and dimeric, respectively. Two newly mononuclear copper(II) complexes, namely, [Cu(L)(bpy)](ClO4) (1) and [Cu(L)(phen)](ClO4) (2) where HL = Schiff base, have been synthesized. UV-visible, EPR and single-crystal X-ray diffraction examinations were used to validate the geometry in solution and solid state. For complex 1, the metal exhibits a coordination sphere between square pyramidal and trigonal bipyramidal geometry (τ, 0.49). A positive CuII/I redox potential indicates a stable switching between CuII and CuI redox states. Despite the monomeric origin, both homogeneous catalysts (1 or 2) in MeOH were found to favor three distinct chemical transformations, namely, ascorbic acid (H2A) to dehydroascorbic acid (DA), benzylamine (Ph-CH2-NH2) to benzaldehyde (Ph-CHO), and 3,5-di-tert-butylcatechol (3,5-DTBC) to 3,5-di-tert-butylquinone (3,5-DTBQ) [kcat: AOase, 9.6 (1) or 2.0 × 106 h-1(2); AmOase, 13.4 (1) or 9.4 × 106 h-1 (2); COase, 2.0 (1) or 1.9 × 103 h-1 (2)]. They exhibit higher levels of AOase activity as indicated by their kcat values compared to the AOase enzyme. The kcat values for COase activity in buffer solution [5.93 (1) or 2.95 × 105 h-1 (2)] are one order lower than those of the enzymes. This is because of the labile nature of the coordinated donor, the flexibility of the ligand, the simplicity of the catalyst-substrate interaction, and the positive CuII/I redox potential. Interestingly, more efficient catalysis is promoted by 1 and 2 concerning that of other mono- and dicopper(II) complexes.
Subject(s)
Amine Oxidase (Copper-Containing) , Ascorbic Acid , Catechol Oxidase , Copper , Catechol Oxidase/chemistry , Catechol Oxidase/metabolism , Ascorbic Acid/chemistry , Copper/chemistry , Amine Oxidase (Copper-Containing)/chemistry , Amine Oxidase (Copper-Containing)/metabolism , Oxidation-Reduction , Coordination Complexes/chemistry , Ascorbate Oxidase/chemistry , Ascorbate Oxidase/metabolism , Biomimetic Materials/chemistry , Biomimetics , Catalysis , Crystallography, X-RayABSTRACT
A sensitive photoelectrochemical (PEC) sensor based on hexagonal carbon nitride tubes (HCNT) as photoactive material was prepared for the detection of human epidermal growth factor receptor 2 (HER2). Magnetic Fe3O4 nanospheres (MNs) modified with anti-HER2 antibodies were employed for highly efficient capture of HER2 from serum sample, and Co3O4 nanoparticles (Co3O4 NPs) modified with ascorbic acid oxidase (AAO) as well as HER2 aptamer were used for signal amplification. When the aptamer-Co3O4-AAO probe was captured onto the electrode surface through the specific binding of the aptamer with HER2, the photocurrent intensity decreased. This was because Co3O4 NPs competed with HCNT for consumption of the excitation energy. As a consequence AAO catalyzed the oxidation of the electron donor (AA), and the aptamer-Co3O4-AAO probe increased the steric hindrance at the electrode surface, leading to significant photocurrent intensity decrease, thus realizing multiple signal amplification. Based on this signal amplification strategy, at 0 V (vs Ag/AgCl), the PEC sensor shows a wide linear response ranging from 1 pg mL-1 to 1 ng mL-1 with a low detection limit of 0.026 pg mL-1 for HER2. Importantly, the prepared PEC sensor was applied for detection of HER2 in human serum samples with recoveries between 98.8 and 101%. Sensitive photoelectrochemical sensor based on Co3O4 nanoparticles modified with ascorbic acid oxidase for signal amplification is reported.
Subject(s)
Ascorbate Oxidase/chemistry , Cobalt/chemistry , Electrochemical Techniques/methods , Oxides/chemistry , Receptor, ErbB-2/blood , Antibodies, Immobilized/immunology , Aptamers, Nucleotide/chemistry , Ascorbic Acid/chemistry , Biosensing Techniques/methods , Enzymes, Immobilized/chemistry , Humans , Immunomagnetic Separation , Limit of Detection , Magnetite Nanoparticles/chemistry , Nanocomposites/chemistry , Photochemical Processes , Receptor, ErbB-2/chemistry , Receptor, ErbB-2/immunology , Reproducibility of ResultsABSTRACT
Nanozymes are artificial enzymes, which can substitute traditional biological enzymes for multifield applications. However, to date, it remains challenging to search novel mimic enzymes or multienzyme mimics. Herein, a facile and green method for preparing monodisperse, homogeneous copper nanoclusters (Cu NCs) with smaller size was developed, which used cysteamine as a template and hydrazine hydrate as a reductant to reduce Cu2+. The as-prepared Cu NCs exhibited excellent tetraenzyme-like activities, including peroxidase (POD)-, catalase (CAT)-, superoxide dismutase (SOD)-, and ascorbic acid oxidase (AAO)-mimic activities. The mechanisms, kinetics, and catalytic performances of Cu NCs were systematically studied. Moreover, based on the POD-like activity of Cu NCs, sensitive and simple colorimetric sensing glutathione (GSH) was explored, with the low limit of detection of 0.89 µM GSH (S/N = 3). Additionally, a novel fluorimetric ascorbic acid (AA) sensor was developed with the linear range of 0.5-30 µM and limit of detection (LOD) of 0.144 µM, on the basis of the principle that AA is oxidized to dehydroascorbic acid (DHAA) specifically catalyzed by the AAO-like activity of Cu NCs, while DHAA can further react with o-phenylenediamine (OPDA) to generate a highly fluorescent quinoxaline (DFQ) derivative. The as-proposed colorimetric GSH sensor and the fluorimetric AA sensor were capable of detecting GSH and AA, respectively, in real samples accurately and reproducibly. Thus, the Cu NCs-based multienzyme mimic is a promising candidate for biocatalysis and biosensing.
Subject(s)
Ascorbic Acid/analysis , Colorimetry , Copper/chemistry , Fluorometry , Glutathione/analysis , Metal Nanoparticles/chemistry , Ascorbate Oxidase/chemistry , Ascorbate Oxidase/metabolism , Ascorbic Acid/metabolism , Catalase/chemistry , Catalase/metabolism , Copper/metabolism , Glutathione/metabolism , Particle Size , Peroxidase/chemistry , Peroxidase/metabolism , Superoxide Dismutase/chemistry , Superoxide Dismutase/metabolism , Surface PropertiesABSTRACT
Ascorbate oxidases are an enzyme group that has not been explored to a large extent. So far, mainly ascorbate oxidases from plants and only a few from fungi have been described. Although ascorbate oxidases belong to the well-studied enzyme family of multi-copper oxidases, their function is still unclear. In this study, Af_AO1, an enzyme from the fungus Aspergillus flavus, was characterized. Sequence analyses and copper content determination demonstrated Af_AO1 to belong to the multi-copper oxidase family. Biochemical characterization and 3D-modeling revealed a similarity to ascorbate oxidases, but also to laccases. Af_AO1 had a 10-fold higher affinity to ascorbic acid (KM = 0.16 ± 0.03 mM) than to ABTS (KM = 1.89 ± 0.12 mM). Furthermore, the best fitting 3D-model was based on the ascorbate oxidase from Cucurbita pepo var. melopepo. The laccase-like activity of Af_AO1 on ABTS (Vmax = 11.56 ± 0.15 µM/min/mg) was, however, not negligible. On the other hand, other typical laccase substrates, such as syringaldezine and guaiacol, were not oxidized by Af_AO1. According to the biochemical and structural characterization, Af_AO1 was classified as ascorbate oxidase with unusual, laccase-like activity.
Subject(s)
Ascorbate Oxidase/metabolism , Aspergillus flavus/enzymology , Laccase/metabolism , Amino Acid Sequence , Ascorbate Oxidase/chemistry , Copper/metabolism , Kinetics , Laccase/chemistry , Models, Molecular , Oxidation-Reduction , Substrate SpecificityABSTRACT
An amperometric L-ascorbic acid biosensor utilizing ascorbate oxidase (AOx) immobilized onto poly(L-aspartic acid) (P(L-Asp)) film was fabricated on carbon nanofiber (CNF) and nanodiamond particle (ND)-modified glassy carbon electrode (GCE). Effects of AOx, ND, and CNF amounts were investigated by monitoring the response currents of the biosensor at different amounts of AOx, ND, and CNF. The electropolymerization step of L-aspartic acid on CNF-ND/GCE surface was also optimized. Scanning electron microscopy (SEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) techniques were used to enlighten the modification steps of the biosensor. The effects of pH and applied potential were studied in detail to achieve the best analytical performance. Under optimized experimental conditions, the AOx/P(L-Asp)/ND-CNF/GCE biosensor showed a linear response to L-ascorbic acid in the range of 2.0 × 10-7-1.8 × 10-3 M with a detection limit of 1.0 × 10-7 M and sensitivity of 105.0 µAmM-1 cm-2. The novel biosensing platform showed good reproducibility and selectivity. The strong interaction between AOx and the P(L-Asp)/ND-CNF matrix was revealed by the high repeatability (3.4%) and good operational stability. The AOx/P(L-Asp)/ND-CNF/GCE biosensor was successfully applied to the determination of L-ascorbic acid in vitamin C effervescent tablet and pharmaceutical powder containing ascorbic acid with good results, which makes it a promising approach for quantification of L-ascorbic acid. Graphical abstract.
Subject(s)
Ascorbate Oxidase/chemistry , Ascorbic Acid/analysis , Biosensing Techniques , Carbon/chemistry , Electrochemical Techniques/instrumentation , Electrodes , Nanodiamonds/chemistry , Nanofibers/chemistry , Limit of Detection , Reproducibility of ResultsABSTRACT
OBJECTIVE: To explore the total phenolic and flavonoid content, enzymatic, non-enzymatic antioxidant properties, anti-inflammation and anticancer activities of hexane, ethyl acetate and methanol extracts of Floscopa scandens (F. scandens). METHODS: Non-enzymatic antioxidant activity was examined by 2, 2-diphenyl-1-picrylhydrazyl assay, nitric oxide scavenging assay, hydroxyl radical scavenging assay, reducing power assay, hydrogen peroxide scavenging assay, superoxide scavenging assay and metal chelating assay. Enzymatic antioxidant ability was screened for the antioxidant enzymes such as ascorbate oxidase, peroxidase, catalase and polyphenol oxidase. The anti-inflammatory property was proved with the inhibition of protein denaturation and protease inhibitory assays. In vitro anticancer activity was assessed by cell viability assay. RESULTS: Methanol extract contained high amount of phenols (198.41 mg catechol equivalent/gram extract) and flavonoids (101.70 mg quercetin equivalent/gram extract) showed higher activity than hexane and ethyl acetate extracts in all experiments. Fresh plant showed considerable enzymatic antioxidant activity. CONCLUSION: The results revealed that the methanol extracts of F. scandens could be used as a potential source of antioxidant, anti-inflammatory and anticancer bioactive compounds.
Subject(s)
Anti-Inflammatory Agents/chemistry , Antineoplastic Agents/chemistry , Antioxidants/chemistry , Commelinaceae/chemistry , Enzyme Inhibitors/chemistry , Plant Extracts/chemistry , Anti-Inflammatory Agents/pharmacology , Antineoplastic Agents/pharmacology , Antioxidants/pharmacology , Ascorbate Oxidase/antagonists & inhibitors , Ascorbate Oxidase/chemistry , Catalase/antagonists & inhibitors , Catalase/chemistry , Cell Line , Cell Survival/drug effects , Flavonoids/chemistry , Flavonoids/pharmacology , Humans , Peroxidase/antagonists & inhibitors , Peroxidase/chemistry , Phenols/chemistry , Phenols/pharmacology , Plant Extracts/pharmacology , Plant Leaves/chemistry , Quercetin/chemistry , Quercetin/pharmacologyABSTRACT
We developed an enzymatic assay system enabling easy quantification of 4-aminobutyric acid (GABA). The reaction of GABA aminotransferase obtained from Streptomyces decoyicus NBRC 13977 was combined to those of the previously developed glutamate assay system using glutamate oxidase and peroxidase. The three-enzyme system allowing GABA-dependent dye formation due to the oxidative coupling between 4-aminoantipyrine and Trinder's reagent enabled accurate quantification of 0.2 - 150 mg/L GABA. A pretreatment mixture consisting of glutamate oxidase, ascorbate oxidase and catalase eliminating glutamate, ascorbate, and hydrogen peroxide, respectively, was also prepared to remove those inhibitory substances from samples. Thus, constructed assay kit was used to measure the GABA content in tomato samples. The results were almost the same as that obtained by the conventional method using liquid chromatography-tandem mass spectrometry. The kit will become a promising tool especially for the on-site measurement of GABA content in agricultural products.
Subject(s)
4-Aminobutyrate Transaminase/chemistry , Amino Acid Oxidoreductases/chemistry , Colorimetry/methods , Enzyme Assays/methods , Peroxidase/chemistry , gamma-Aminobutyric Acid/analysis , Ampyrone/chemistry , Ascorbate Oxidase/chemistry , Catalase/chemistry , Chromatography, Liquid , Enzyme Assays/economics , Ferric Compounds/chemistry , Glutamic Acid/chemistry , Hydrogen Peroxide/chemistry , Solanum lycopersicum/chemistry , Oxidative Coupling , Recombinant Proteins , Streptomyces/enzymology , Tandem Mass SpectrometryABSTRACT
Although oxidase mimetic nanozymes have been widely investigated, specific biological molecules have rarely been explored as substrates, particularly in the case of ascorbate oxidase (AAO) mimetic nanozymes. Herein, we demonstrate for the first time that copper(II) oxide nanoparticles (CuO NPs) catalyze the oxidation of ascorbic acid (AA) by dissolved O2 (as a green oxidant) to form dehydroascorbic acid (DHAA), thus functioning as a new kind of AAO mimic. Under neutral conditions, the Michaelis-Menten constant of CuO NPs (0.1302â mm) is similar to that of AAO (0.0840â mm). Furthermore, the robustness of CuO NPs is greater than that of AAO, thus making them suitable for applications under various conditions. As a demonstration, a fluorescence AA sensor based on the AAO mimetic activity of CuO NPs was developed. To obtain a fluorescent product, o-phenylenediamine (OPDA) was used to react with the DHAA produced by the oxidation of AA catalyzed by CuO NPs. The developed sensor was cost-effective and easy to fabricate and exhibited high selectivity/sensitivity with a wide linear range (1.25×10-6 to 1.125×10-4 m) and a low detection limit (3.2×10-8 m). The results are expected to aid in expanding the applicability of oxidase mimetic nanozymes in a variety of fields such as biology, medicine, and detection science.
Subject(s)
Biomimetic Materials/metabolism , Copper/chemistry , Metal Nanoparticles/chemistry , Ascorbate Oxidase/chemistry , Ascorbate Oxidase/metabolism , Ascorbic Acid/chemistry , Ascorbic Acid/metabolism , Biomimetic Materials/chemistry , Catalysis , Kinetics , Oxidation-Reduction , Oxygen/chemistry , Oxygen/metabolismABSTRACT
We studied the laccase-catalysed oxygenation of methoxyphenolic food ingredients, such as 2-methoxyphenol (guaiacol) and 2,6-dimethoxyphenol (syringol), isomers such as 3- and 4-methoxyphenol, and 2,3-, 3,4- and 3,5-dimethoxyphenol. These methoxyphenolic substrates generate unstable free radicals, which leads to the erroneous determination of steady state rates. The addition of small quantities of ascorbic acid as coupling reagent generates a lag period because it reduces free radicals to methoxyphenols. Measurement of the length of the lag period provides the reliable determination of true steady state rates. We describe the application of this chronometric method to the kinetic characterization of the oxidation of the above methoxyphenolic substrates by Trametes versicolor laccase.
Subject(s)
Enzyme Assays/methods , Food Ingredients/analysis , Laccase/chemistry , Phenols/analysis , Ascorbate Oxidase/chemistry , Enzyme Activation , Hydrogen-Ion Concentration , Isomerism , Kinetics , Molecular Structure , Spectrum Analysis , Substrate SpecificityABSTRACT
A simple enzyme-based nanohybrid material was fabricated via immobilizing ascorbic acid oxidase (AO) on the surface of flower-like electrodeposited gold nanoparticles (dpAu) and reduced graphene oxide (rGO) modified glassy carbon electrodes (GCEs). The composite material was used for stereoselective interaction with ascorbic acid (AA) and isoascorbic acid (IAA). Herein, AO was applied as a stereoselective selector, and the dpAu/rGO nanohybrid not only acted as a supporter for high loading of AO, but also served as the nanomaterial for signal amplification. The results showed obvious peak current differences between AA and IAA, indicating that this strategy could be employed to recognize AA and IAA. Under the optimum conditions, the sensor exhibited a good linear response to AA and IAA in a linear range of 1.0 × 10-4 - 5.0 × 10-3 M. This approach with the merits of simplicity and rapid response provided a promising perspective for identification of AA and IAA.
Subject(s)
Ascorbate Oxidase/metabolism , Ascorbic Acid/analysis , Biosensing Techniques/methods , Ascorbate Oxidase/chemistry , Electrochemistry , Electroplating , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Gold/chemistry , Graphite/chemistry , Metal Nanoparticles/chemistry , Models, Molecular , Molecular Conformation , Oxides/chemistry , Stereoisomerism , Substrate SpecificityABSTRACT
An ascorbic acid oxidase (AAO)-ascorbic acid bioevent-based electron donor consumption mode was introduced into the PEC bioassay for the first time. Ternary hybrid bismuth sulfide/silver sulfide/TiO2 nanotube arrays as the photoelectrode coupled with AAO attached to SiO2 as a dual signal quenching strategy were employed for sensitivity enhancement.
Subject(s)
Ascorbate Oxidase/chemistry , Ascorbic Acid/chemistry , Biosensing Techniques , Electrochemical Techniques , Electrons , Nanotechnology , Ascorbate Oxidase/metabolism , Ascorbic Acid/metabolism , Bismuth/chemistry , Nanotubes/chemistry , Photochemical Processes , Silver Compounds/chemistry , Sulfides/chemistry , Titanium/chemistryABSTRACT
A new colorimetric immunosensing platform accompanying enzyme cascade amplification strategy was fabricated for quantitative screening of small-molecular mycotoxins (aflatoxin B1, AFB1 used in this case) coupling with enzyme-controlled dissolution of MnO2 nanoflakes. The visual colored assay was executed by high-efficient MnO2-3,3',5,5'-tetramethylbenzidine (TMB) system (blue). In the presence of ascorbic acid, MnO2 nanoflakes were dissolved into Mn2+ ions, thus resulting in a perceptible color change from blue to colorless. The reaction could be weakened through ascorbate oxidase to catalyze ascorbic acid into dehydroascorbic acid, which indirectly depended on the concentration of ascorbate oxidase. By using ascorbate oxidase/ anti-AFB1 antibody-labeled gold nanoparticles, a novel competitive-type colorimetric enzyme immunoassay was developed for detection of AFB1 on AFB1-bovine serum albumin (BSA)-conjugated magnetic beads. Upon addition of target AFB1, the analyte competed with the conjugated AFB1-BSA on the magnetic beads for the labeled anti-AFB1 antibody on the gold nanoparticles. Under optimal conditions, the absorbance decreased with increasing target AFB1 within the dynamic range of 0.05-150ngmL-1 with a detection limit of 6.5pgmL-1 at the 3Sblank level. The precision and specificity of the MnO2-TMB-based immunosensing system were acceptable. In addition, method accuracy was further validated for monitoring spiked peanut samples, giving results matched well with those obtained from commercialized AFB1 ELISA kit.
Subject(s)
Aflatoxin B1/analysis , Arachis/microbiology , Biosensing Techniques/methods , Food Contamination/analysis , Manganese Compounds/chemistry , Nanostructures/chemistry , Oxides/chemistry , Animals , Antibodies, Immobilized/chemistry , Arachis/chemistry , Ascorbate Oxidase/chemistry , Aspergillus flavus/chemistry , Benzidines/chemistry , Cattle , Colorimetry/methods , Food Microbiology , Gold/chemistry , Immunoassay/methods , Limit of Detection , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Nanostructures/ultrastructure , Serum Albumin, Bovine/chemistry , SolubilityABSTRACT
Apoplastic ascorbate oxidase (AO) plays significant roles in plant cell growth. However, the mechanism of underlying the transcriptional regulation of AO in Gossypium hirsutum remains unclear. Here, we obtained a 1,920-bp promoter sequence from the Gossypium hirsutum ascorbate oxidase (GhAO1) gene, and this GhAO1 promoter included a number of known cis-elements. Promoter activity analysis in overexpressing pGhAO1::GFP-GUS tobacco (Nicotiana benthamiana) showed that the GhAO1 promoter exhibited high activity, driving strong reporter gene expression in tobacco trichomes, leaves and roots. Promoter 5'-deletion analysis demonstrated that truncated GhAO1 promoters with serial 5'-end deletions had different GUS activities. A 360-bp fragment was sufficient to activate GUS expression. The P-1040 region had less GUS activity than the P-720 region, suggesting that the 320-bp region from nucleotide -720 to -1040 might include a cis-element acting as a silencer. Interestingly, an auxin-responsive cis-acting element (TGA-element) was uncovered in the promoter. To analyze the function of the TGA-element, tobacco leaves transformed with promoters with different 5' truncations were treated with indole-3-acetic acid (IAA). Tobacco leaves transformed with the promoter regions containing the TGA-element showed significantly increased GUS activity after IAA treatment, implying that the fragment spanning nucleotides -1760 to -1600 (which includes the TGA-element) might be a key component for IAA responsiveness. Analyses of the AO promoter region and AO expression pattern in Gossypium arboreum (Ga, diploid cotton with an AA genome), Gossypium raimondii (Gr, diploid cotton with a DD genome) and Gossypium hirsutum (Gh, tetraploid cotton with an AADD genome) indicated that AO promoter activation and AO transcription were detected together only in D genome/sub-genome (Gr and Gh) cotton. Taken together, these results suggest that the 1,920-bp GhAO1 promoter is a functional sequence with a potential effect on fiber cell development, mediated by TGA-element containing sequences, via the auxin-signaling pathway.
Subject(s)
Ascorbate Oxidase/genetics , Gossypium/genetics , Indoleacetic Acids/metabolism , Promoter Regions, Genetic/genetics , Ascorbate Oxidase/biosynthesis , Ascorbate Oxidase/chemistry , Ascorbate Oxidase/isolation & purification , Cloning, Molecular , Gene Expression Regulation, Enzymologic , Gossypium/enzymology , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , Signal Transduction/genetics , Nicotiana/enzymology , Nicotiana/geneticsABSTRACT
Quantitative analysis of antioxidants in a fast, simple and accurate manner is of great importance in the view of real-time monitoring the health of individuals. Recently, we have developed a UV/vis spectroscopic microfluidic sensor to specifically quantify ascorbic acid based on the immobilization of ascorbate oxidase, a relatively unstable enzyme. In this work, three different strategies for the immobilization of the unstable enzyme, including alumina sol-gel encapsulation, physisorption to PDMS channels with, and without alumina xerogel modification, were compared to build a microsensor. We found that the loading amount of the enzyme is not the determinative factor for the performance of the microfluidic biosensor but the retained activity of the enzyme and diffusion in the microfluidic channel. Taking into account of the two factors, the protocol of adsorbing enzymes to alumina (Al2O3) xerogel modified PDMS surface was demonstrated to be the best for preparing the microfluidic sensor among the utilized protocols. The microsensor prepared under the optimized protocol was further used to quantify ascorbic acid in human blood, where only dozens of microliters of blood (few drops) was required, demonstrating its potential application in clinical diagnosis. The developed strategy is featured with optimized enzymatic activity, simple process of microfluidic platform, low sample consumption, and straightforward spectrophotometry based detection.
Subject(s)
Ascorbic Acid/blood , Biosensing Techniques/methods , Microfluidic Analytical Techniques/methods , Spectrophotometry, Ultraviolet/methods , Adsorption , Aluminum Oxide/chemistry , Ascorbate Oxidase/chemistry , Ascorbate Oxidase/metabolism , Ascorbic Acid/metabolism , Dimethylpolysiloxanes/chemistry , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Humans , Limit of Detection , Phase TransitionABSTRACT
Creatinine is one of many markers used to investigate kidney function. This paper describes a low-cost enzymatic paper-based analytical device (enz-PAD) for determining urine creatinine. The disposable dead volumes of creatinine enzyme reagents from an automatic analyser cassette were utilised. Whatman No. 3 paper was cut into long rectangular shapes (4×40 mm(2)) on which the enzyme reagents, R1 and R2, were adsorbed in two consecutive regions. The assay was performed by immersing test strips into urine samples contained in microwells to allow creatinine in the sample to react with immobilised active ingredients and, then, traverse via capillary action to the detection area where chromogen products accumulated. The method is based on hydrogen peroxide (H2O2) formation via creatinine conversion using creatininase, creatinase, and sarcosine oxidase. The liberated H2O2 reacts with 4-aminophenazone and 2,4,6-triiodo-3-hydroxybenzoic acid to form quinoneimine with a pink-red colour at the detection zone. The linear range of the creatinine assay was 2.5-25 mg dL(-1) (r(2)=0.983), and the detection limit was 2.0 mg dL(-1). The colorimetric enz-PAD for the creatinine assay was highly correlated with a conventional alkaline picrate method when real urine samples were evaluated (r(2)=0.977; n=40). This simple and nearly zero-cost paper-based device provides a novel alternative method for screening urinary creatinine and will be highly beneficial for developing countries.
Subject(s)
Creatinine/urine , Enzyme Assays/methods , Paper , Ascorbate Oxidase/chemistry , Catalase/chemistry , Colorimetry/methods , Costs and Cost Analysis , Creatinine/chemistry , Enzyme Assays/economics , Humans , Peroxidase/chemistry , Sarcosine Oxidase/chemistry , Ureohydrolases/chemistryABSTRACT
Examples of proteins that incorporate one or more metal ions within their structure are found within a broad range of classes, including oxidases, oxidoreductases, reductases, proteases, proton transport proteins, electron transfer/transport proteins, storage proteins, lyases, rusticyanins, metallochaperones, sporulation proteins, hydrolases, endopeptidases, luminescent proteins, iron transport proteins, oxygen storage/transport proteins, calcium binding proteins, and monooxygenases. The metal coordination environment therein is often generated from residues inherent to the protein, small exogenous molecules (e.g., aqua ligands) and/or macrocyclic porphyrin units found, for example, in hemoglobin, myoglobin, cytochrome C, cytochrome C oxidase, and vitamin B12. Thus, there continues to be considerable interest in employing macrocyclic metal complexes to construct low-molecular weight models for metallobiosites that mirror essential features of the coordination environment of a bound metal ion without inclusion of the surrounding protein framework. Herein, we review and appraise our research exploring the application of the metal complexes formed by two macrocyclic ligands, 1,4,7-triazacyclononane (tacn) and 1,4,7,10-tetraazacyclododecane (cyclen), and their derivatives in biological inorganic chemistry. Taking advantage of the kinetic inertness and thermodynamic stability of their metal complexes, these macrocyclic scaffolds have been employed in the development of models that aid the understanding of metal ion-binding natural systems, and complexes with potential applications in biomolecule sensing, diagnosis, and therapy. In particular, the focus has been on "coordinatively unsaturated" metal complexes that incorporate a kinetically inert and stable metal-ligand moiety, but which also contain one or more weakly bound ligands, allowing for the reversible binding of guest molecules via the formation and dissociation of coordinate bonds. With regards to mimicking metallobiosites, examples are presented from our work on tacn-based complexes developed as simplified structural models for multimetallic enzyme sites. In particular, structural comparisons are made between multinuclear copper(II) complexes formed by such ligands and multicopper enzymes featuring type-2 and type-3 copper centers, such as ascorbate oxidase (AO) and laccase (Lc). Likewise, with the aid of relevant examples, we highlight the importance of cooperativity between either multiple metal centers or a metal center and a proximal auxiliary unit appended to the macrocyclic ligand in achieving efficient phosphate ester cleavage. Finally, the critical importance of the Zn(II)-imido and Zn(II)-phosphate interactions in Zn-cyclen-based systems for delivering highly sensitive electrochemical and fluorescent chemosensors is also showcased. The Account additionally highlights some of the factors that limit the performance of these synthetic nucleases and the practical application of the biosensors, and then identifies some avenues for the development of more effective macrocyclic constructs in the future.
Subject(s)
Biocompatible Materials/chemistry , Coordination Complexes/chemistry , Metals/chemistry , Ascorbate Oxidase/chemistry , Ascorbate Oxidase/metabolism , Biocompatible Materials/metabolism , Biosensing Techniques , Coordination Complexes/metabolism , Cyclams , Heterocyclic Compounds/chemistry , Laccase/chemistry , Laccase/metabolism , Ligands , Molecular Conformation , Ribonucleases/chemistry , Ribonucleases/metabolismABSTRACT
A new carbon ascorbate oxidase-based sensor-biosensor system (SB) was coupled to a dual-channel telemetric device for online simultaneous electrochemical detection of ascorbic acid (AA) and antioxidant capacity in Hamlin, Sanguinello, and Moro orange varieties. The electrocatalytic performances of the SB were investigated by cyclic voltammetry and amperometric techniques. The phenol composition of orange juice of each variety, and the cyclic voltammetries of the most represented phenols, were provided. The in vitro calibrations were performed in PBS (pH 5.6), applying a constant potential of +500 mV. A standard mixture of phenols, based on orange juice composition, was used as reference material for studying SB behavior. SB works at an applied potential of +500 mV, in a concentration range comprised between the LOD 0.26 µM and 20 µM. In this concentration range, limiting the data acquisition time to 2 min, the problems of electrode passivation due to phenols polymerization were overcome. AA calibration showed that the biosensor registered statistically lower currents than the sensor since the enzyme oxidized AA before it reached the electrode surface. Standard mixture calibration showed that currents registered by sensor and biosensor did not statistically differ. The difference between sensor and biosensor AA registered currents was used to calculate an AA selectivity index and, consequently, to determine the AA content and the antioxidant capacity in the juices. The novelty of the SB is its ability to distinguish between AA and phenols contribution to antioxidant capacity. The obtained results were in accordance with reference methods.
Subject(s)
Antioxidants/analysis , Ascorbate Oxidase/metabolism , Ascorbic Acid/analysis , Beverages/analysis , Biosensing Techniques/methods , Food Technology/instrumentation , Food Technology/methods , Ascorbate Oxidase/chemistry , Biosensing Techniques/instrumentation , Electrochemical Techniques , Electrodes , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Oxidation-Reduction , Phenols/analysis , Telemetry/instrumentationABSTRACT
A direct electrochemistry and intramolecular electron transfer of multicopper oxidases are of a great importance for the fabrication of these enzyme-based bioelectrochemical-devices. Ascorbate oxidase from Acremonium sp. (ASOM) has been successfully immobilized via a chemisorptive interaction on the l-cysteine self-assembled monolayer modified gold electrode (cys-SAM/AuE). Thermodynamics and kinetics of adsorption of ASOM on the cys-SAM/AuE were studied using cyclic voltammetry. A well-defined redox wave centered at 166±3mV (vs. AgâAgClâKCl(sat.)) was observed in 5.0mM phosphate buffer solution (pH7.0) at the fabricated ASOM electrode, abbreviated as ASOM/cys-SAM/AuE, confirming a direct electrochemistry, i.e., a direct electron transfer (DET) between ASOM and cys-SAM/AuE. The direct electrochemistry of ASOM was further confirmed by taking into account the chemical oxidation of ascorbic acid (AA) by O2 via an intramolecular electron transfer in the ASOM as well as the electrocatalytic oxidation of AA at the ASOM/cys-SAM/AuE. Thermodynamics and kinetics of the adsorption of ASOM on the cys-SAM/AuE have been elaborated along with its direct electron transfer at the modified electrodes on the basis of its intramolecular electron transfer and electrocatalytic activity towards ascorbic acid oxidation and O2 reduction. ASOM saturated surface area was obtained as 2.41×10(-11)molcm(-2) with the apparent adsorption coefficient of 1.63×10(6)Lmol(-1). The ASOM confined on the cys-SAM/AuE possesses its essential enzymatic function.
Subject(s)
Ascorbate Oxidase/chemistry , Cysteine/chemistry , Enzymes, Immobilized/chemistry , Gold/chemistry , Acremonium/enzymology , Adsorption , Ascorbate Oxidase/metabolism , Biocatalysis , Electrochemistry , Electrodes , Electron Transport , Enzymes, Immobilized/metabolism , Hydrogen-Ion Concentration , Kinetics , ThermodynamicsABSTRACT
Herein, a simple and rapid method is described for detection of L-ascorbic acid by ascorbate oxidase immobilized onto polycarbonate strip pre-activated by 1-fluoro-2-nitro-4-azidobenzene in photochemical reaction. Covalent attachment of ascorbate oxidase was confirmed by XPS studies. The immobilized-ascorbate oxidase shows higher pH, thermal and storage stability in comparison to free enzyme.