Covalent immobilization of α-amylase on hollow metal organic framework coated magnetic phase-change microcapsules for the improvement of its thermostability.

Exploring efficient immobilized carrier for α-Amylase (α-Amy) to enhance its thermostability has significant influence in starch related industry. Here, hollow ZIF-8 (HZIF-8) with amino groups coated magnetic phase change microcapsules (PCM) was designed for covalent immobilization of α-Amy. Magnetic PCM consisting n-docosane core and SiO2/Fe3O4 hybrid shell were firstly synthesized. Then, HZIF-8 shell with amino groups was coated and α-Amy was subsequently immobilized through covalent cross-linking strategy. The morphology, chemical structure and magnetic property of PCM@HZIF-8@α-Amy (PCMHA) were comprehensively characterized. Moreover, heat control property of PCMHA was studied with encapsulation efficiency and thermal energy-storage efficiency of 50.55 % and 50.59 %, respectively. Catalytic activity of immobilized α-Amy was fully investigated with Km and Vmax of 2.773 mg/mL and 1.853 µmol/mL·min, respectively. From reusability and storage stability study, immobilized α-Amy not only maintained rather high activity after 9 cycles' reuse, but also exhibited good activity under high salt ion condition after 7 days.

Whey protein isolate and inulin-glycosylated conjugate affect the physicochemical properties and oxidative stability of pomegranate seed oil emulsion.

Glycosylated protein was obtained by the reaction of whey protein isolate(WPI) with inulin of different polymerization degrees and was used to stabilize a pomegranate seed oil emulsion. The physicochemical and antioxidative properties of the emulsions were assessed, and the impacts of accelerated oxidation on pomegranate seed oil were examined. The interfacial tension of WPI and short-chain inulin (SCI)-glycosylated conjugate (WPI-SCI) gradually decreased with increasing glycosylation reaction time. Emulsions stabilized by WPI-SCI (72 h) were the most stable, with a thick interfacial film on the surface of the droplets. After accelerated oxidation for 72 h, WPI-SCI inhibited the oxidation of oil in the emulsion. GC-IMS results showed that the production of harmful volatile components in oil was inhibited, and the peroxide strength was less than 30 mmol/kg oil. This study contributes to understanding of stable storage of lipids.

Pd nanoparticles decorated ultrathin 2D metal-organic framework nanosheets with enhanced peroxidase-mimic activity and colorimetric assay of glucose.

In addition to size, shape and morphology, enzyme-mimetic property could be efficiently regulated by controlling composition, forming complexes or hybrids, and surface modification. Herein, Pd nanoparticles with an average diameter of 2.52 nm were decorated on ultrathin 2D copper(ii)-porphyrin derived metal-organic framework (MOF) nanosheets by a simple reduction method for catalytic activity regulation. In comparison with other nanozymes, the as-synthesized Pd modified 2D MOF hybrid nanosheets (Pd@Cu-TCPP(Fe)) presented excellent peroxidase-mimic activity, exhibiting an even superior catalytic ability towards H2O2 with a Michaelis-Menten constant as low as 2.33 mM. Based on a cascade reaction between glucose oxidase and Pd@Cu-TCPP(Fe), a colorimetric method for the detection of glucose was established and validated with a wide linear range (0.2-8.0 mM), good recovery (89.5-94.2%) and nice reproducibility (3.65%). All these features guaranteed its excellent ability for glucose determination in human cerebrospinal fluids. This study could offer a valuable reference for constructing novel optical biosensors.

Two dimensional iron metal-organic framework nanosheet with peroxidase-mimicking activity for colorimetric detection of hypoxanthine related to shrimp freshness.

Two dimensional iron metal-organic framework nanosheet (2D Fe MOF) was facilely synthesized at room temperature by simple stirring of iron salts and terephthalic acid ligand in a mixed solution containing triethylamine. Its morphology and structure were fully characterized by TEM, AFM, XPS and TEM element mapping. Then, its peroxidase-mimicking activity was studied by using H2O2 and 3, 3', 5, 5'- tetramethylbenzidine as substrate. Km and Vmax of 2D Fe MOF towards H2O2 were 0.02 mM and 2.08 × 10-8 M s-1, respectively. Through the formation of cascade reaction between xanthine oxidase and 2D Fe MOF, a visual method for hypoxanthine (Hx) detection was constructed to evaluate aquatic products freshness. After effective validation, this method presented wide linear range (5.0-500.0 µM), low limit of detection (3.29 µM), satisfied accuracy (recovery of 94.78-99.85%), and good selectivity. By using this method, Hx content in shrimp samples at different storage time were determined.

Preparation, Characterization and Antioxidant Activity of Glycosylated Whey Protein Isolate/Proanthocyanidin Compounds.

A glycosylated protein/procyanidin complex was prepared by self-assembly of glycosylated whey protein isolate and proanthocyanidins (PCs). The complex was characterized through endogenous fluorescence spectroscopy, polyacrylamide gel electrophoresis, Fourier infrared spectroscopy, oil-water interfacial tension, and transmission electron microscopy. The results showed that the degree of protein aggregation could be regulated by controlling the added amount of procyanidin, and the main interaction force between glycosylated protein and PCs was hydrogen bonding or hydrophobic interaction. The optimal binding ratio of protein:PCs was 1:1 (w/w), and the solution pH was 6.0. The resulting glycosylated protein/PC compounds had a particle size of about 119 nm. They exhibited excellent antioxidant and free radical-scavenging abilities. Moreover, the thermal denaturation temperature rose to 113.33 °C. Confocal laser scanning microscopy (CLSM) images show that the emulsion maintains a thick interface layer and improves oxidation resistance with the addition of PCs, increasing the application potential in the functional food industry.

Improving the physicochemical stability of Pickering emulsion stabilized by glycosylated whey protein isolate/cyanidin-3-glucoside to deliver curcumin.

Protein-polysaccharide-polyphenol delivery systems function as a promising tool to deliver bioactive ingredients aiming to improve their solubility and bioavailability. In this study, whey protein isolate (WPI), short-chain inulin (SCI), and cyanidin-3-glucoside (C3G) were first used to stabilize Pickering emulsions. The physicochemical properties and stability of curcumin encapsulated or not in Pickering emulsions were explored. Results showed that glycosylation and C3G reduced surface and interfacial tension on protein surfaces and inhibited the aggregation of emulsion droplets, thereby reducing the emulsion's particle size. WPI-SCI/C3G stabilized Pickering emulsion had the best stability. The CLSM results showed that the WPI-SCI and WPI-SCI/C3G stabilized emulsions were uniformly dispersed, suggesting that glycosylation and the interaction between protein and C3G enhanced the adsorption capacity of the interfacial protein and improved the stability of the Pickering emulsion. The retention rates of curcumin-loaded WPI-SCI- (67.34 %) and WPI-SCI/C3G- (77.07 %) stabilized Pickering emulsions on day 8 of storage were higher than those in WPI- (33.97 %) and WPI/C3G- (37.02 %) stabilized emulsions, and the degradation half-life was also extended from 7 days to >15 days. These findings provide a theoretical basis for the application of WPI Pickering emulsion and indicate a useful means for the delivery of bioactive components.

Metalloporphyrin and gold nanoparticles modified hollow zeolite imidazole Framework-8 with excellent peroxidase like activity for quick colorimetric determination of choline in infant formula milk powder.

Metalloporphyrin and gold nanoparticles (AuNPs) were fixed on the surface of hollow zeolite imidazole framework-8 (HZIF-8) by cation exchange and cross-linking reaction. The obtained hybrid nanozyme, Au/HZIF-8@TCPP(Fe), was fully characterized by TEM, HRTEM, EDS element mapping and XPS. Then, its peroxidase-like activity was explored with Km of 1.74 mM and Vmax of 9.60 × 10-8 M·S-1 towards H2O2, indicating excellent catalytic activity. Based on cascade reaction between choline oxidase and Au/HZIF-8@TCPP(Fe), a quick colorimetric method was established for choline detection in infant formula milk powder. After comprehensive verification, this method presented the merits of simple operation, satisfied detection limit (0.05 mM), wide linear range (0.05-2.0 mM), high accuracy (recovery of 92.2%-105.2%) and nice selectivity. This colorimetric method was applied to the determination of choline in milk powders of five brands. Our study could offer valuable reference for finding highly efficient nanozyme and constructing novel optical biosensors.

Colorimetric quantification of sodium benzoate in food by using d-amino acid oxidase and 2D metal organic framework nanosheets mediated cascade enzyme reactions.

A rapid colorimetric method for detecting sodium benzoate in food products was established based on the d-amino acid oxidase (DAAO) and 2D metal organic framework (2D MOF) nanosheets mediated cascade enzyme reactions. Firstly, the synthesized 2D MOF nanosheets served as high efficient nanozyme with outstanding peroxidase-like catalytic activity and catalyzed the color reaction between H2O2 and 3, 3', 5, 5'- tetramethylbenzidine. Secondly, sodium benzoate as a competitive inhibitor of DAAO, could influence the production of H2O2 in DAAO mediated oxidation reaction. After a combination of those two reactions, this colorimetric quantitative method was constructed and validated for sodium benzoate determination with wide linear range (2.0-200.0 µM), low limit of detection (2.0 µM), high accuracy (recovery rate in 95.80-108.00%) and satisfied selectivity. Lastly, this method was utilized to analyze sodium benzoate concentration in juice, wine and vinegar by naked eyes.

Ultrasmall Au nanoparticles modified 2D metalloporphyrinic metal-organic framework nanosheets with high peroxidase-like activity for colorimetric detection of organophosphorus pesticides.

Ultrasmall Au nanoparticles (UsAuNPs) in the size range of 4.0-7.0 nm was successfully immobilized on the surface of 2D metalloporphyrinic metal-organic framework nanosheets (2D MOF). Firstly, The obtained hybrid nanomaterial, UsAuNPs/2D MOF, was fully characterized by TEM, HRTEM, element mapping images and XPS. Then, the peroxidase-like activity of UsAuNPs/2D MOF was comparatively studied with other hybrid nanozyme to explore the influence of AuNPs size on peroxidase-like activity. Further, UsAuNPs/2D MOF with outstanding peroxidase-like activity was selected to form ternary cascade enzyme reaction with acetylcholinesterase (AChE) and choline oxidase (ChOx). Based on the inhibitory effect of organophosphorus pesticides on AChE, a fast and sensitive colorimetric method was established for trichlorfon detection with the advantages of simple operation, low detection limit (1.7 µM), good linear range (1.7-42.4 µM) and high accuracy (recovery rate of 96.6-105.3%). Finally, this method was applied to visual analysis of trichlorfon concentration in tomatoes, cucumbers and eggplants.

Comparative research on the metabolism of metoprolol by four CYP2D6 allelic variants in vitro with LC-MS/MS.

Specific study about the effect of cytochrome P450 2D6 (CYP2D6) polymorphisms on the metabolism of clinic drugs is of great significance for drug safety investigation. Here, the interaction between CYP2D6 variants (*1, *2, *10, *39) and metoprolol (MET) was intensively researched in vitro from the aspect of drug-enzyme kinetic study. To obtain quantitative data, α-hydroxymetoprolol (main metabolite of MET) was selected as an ideal analyte and an LC-MS/MS method was adopted for sample determination. Firstly, by selecting suitable internal standard and optimizing separation condition, the LC-MS/MS method was established and validated. Then, the drug-enzyme incubation system was optimized by two parameters: incubation time and amount of enzyme. Lastly, the interaction between CYP2D6 allelic variants and MET was characterized by Km, Vmax and CLint. As a result, four CYP2D6 enzymes displayed diverse Km or Vmax towards MET and the values of CLint showed a wide range from 8.91 to 100%. Relative to CYP2D6*1 (CLint*1 = 100%), CYP2D6*2 demonstrated the second high catalytic activity (CLint*2/*1 = 74.87%) while CYP2D6*39 (CLint*39/*1 = 29.65%) and CYP2D6*10 (CLint*10/*1 = 8.91%) showed minimal catalytic activity. This comprehensive in vitro data suggested the prominent influence of CYP2D6 polymorphisms on the metabolism of MET, which could offer valuable information for personalized administration of MET in clinic.

Comparative study on the interaction between 3 CYP2C9 allelic isoforms and benzbromarone by using LC-MS/MS method.

Benzbromarone is a uricosuric drug metabolized predominantly by cytochrome P450 2C9 from in vitro findings. Human CYP2C9 exhibits extensive genetic polymorphism and numbers of clinic studies have demonstrated that CYP2C9 genetic polymorphism has a significant influence on the pharmacokinetics of benzbromarone. But in vitro study on the interaction between CYP2C9 allelic isoforms and benzbromarone was rare. Here, an LC-MS/MS method was established and validated to determine the concentration of benzbromarone in different CYP2C9 enzyme incubation systems for the drug-enzyme interaction study. By selecting appropriate internal standard and optimizing separation system, including mobile phase, sample solvent and gradient elution condition, this LC-MS/MS method was developed with fine linearity (r2≥0.996), good reproducibility (RSD≤6.6%), high stability (92.37-114.67%), efficient recovery (91.23-109.82%) and acceptable matrix effect (110.54-115.31%). Based on this method, the interaction between 3 CYP2C9 allelic isoforms and benzbromarone was researched by kinetics parameters (Km, Vmax, Clint). As a result, CYP2C9*1 displayed the highest metabolic activity towards benzbromarone, CYP2C9*2 showed a little lower catalytic activity than CYP2C9*1 (relative clearance/*1=85.86%), CYP2C9*3 showed the lowest catalytic activity (relative clearance/*1=21.57%). The result illustrated that various CYP2C9 allelic isoforms showed different enzymatic activities towards benzbromarone, which could offer effective consultation for personalized administration in clinic.

Influence of ionic liquids as electrolyte additives on chiral separation of dansylated amino acids by using Zn(II) complex mediated chiral ligand exchange CE.

In this work, investigation of the comparative influence of diverse ionic liquids (ILs) as electrolyte additives on the chiral separation of dansylated amino acids by using Zn(II)-L-arginine complex mediated chiral ligand exchange CE (CLE-CE) was conducted. It has been found that not only the varied substituted group number, but also the alkyl chain length of the substituted group on imidazole ring in the structure of ILs show different influence on chiral separation of the analytes in the CLE-CE system, which could be understood by their direct influence on EOF. Meanwhile, the variation of anion in the structure of ILs displayed remarkably changed performance and the ILs with Cl(-) showed the most obvious promoting effect on the chiral separation performance. Among the investigated seven ILs, 1-butyl-3-methylimidazolium chloride was validated to be the proper electrolyte additive in the CLE-CE system. Moreover, it has been observed that 1-butyl-3-methylimidazolium chloride also has obvious promotive effect on the labeling performance. The results have demonstrated that the ILs with different structures have important relation to their performance in CLE-CE and to their labeling efficiency in dansylation of the analytes.

L-Lysine-derived ionic liquids as chiral ligands of Zn(II) complexes used in ligand-exchange CE.

Amino acid ionic liquids (AAILs) with L-lysine (L-Lys) as anion were synthesized and applied as new chiral ligands in Zn(II) complexes for chiral ligand-exchange CE. After effective optimization, baseline enantioseparation of seven pairs of dansylated amino acids was achieved with a buffer of 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4 , and 6.0 mM [C6 mim][L-Lys] at pH 8.2. To validate the unique behavior of AAILs, a comparative study between the performance of Zn(II)-L-Lys and Zn(II)-[C6 mim][L-Lys] systems was conducted. In Zn(II)-[C6 mim][L-Lys] system, it has been found that the improved chiral resolution could be obtained and the migration times of the three test samples were markedly prolonged. Then the separation mechanism was further discussed. The role of [C6 mim][L-Lys] indicated clearly that the synthesized AAILs could be used as chiral ligands and would have potential utilization in separation science in future.

A novel chiral ligand exchange capillary electrophoresis system with amino acid ionic liquid as ligand and its application in screening D-amino-acid oxidase inhibitors.

A novel amino acid ionic liquid (AAIL) with L-ornithine (L-Orn) as anion was successfully synthesized, and subsequently applied as an available chiral ligand coordinated with Zn(II) in a chiral ligand exchange capillary electrophoresis (CLE-CE) system for the enantioseparation of dansyl amino acids (Dns-D,L-AAs). The influence of key parameters, such as buffer pH, concentration ratio of Zn(II) to ligand and complex concentration, was investigated in detail. Eleven pairs of Dns-D,L-AAs enantiomers were baseline separated and three pairs were partly separated under the optimum conditions. For exploring its potential application, the quantitative features of this proposed method were studied. Good linearity (r(2) = 0.999) and favorable repeatability (RSD ≤ 3.4%) were obtained by using Dns-D,L-Met as the test analyte. Finally, this method was employed to investigate the inhibition efficiency of d-amino acid oxidase (DAAO) inhibitors, which may pave a new way for the high-throughput screening of enzyme inhibitors and relevant drug discovery.

Ionic liquids with amino acids as cations: novel chiral ligands in chiral ligand-exchange capillary electrophoresis.

Ionic liquids (ILs) with L-proline (L-Pro) as cations have been developed for the novel chiral ligands coordinated with Cu(II) in chiral ligand exchange capillary electrophoresis (CLE-CE). Four kinds of amino acid ionic liquids (AAILs), including [L-Pro][CF(3)COO], [L-Pro][NO(3)], [L-Pro][BF(4)] and [L-Pro(2)][SO(4)], were successfully synthesized. Among them, [L-Pro][CF(3)COO] was selected as the model ligand to optimize the separation conditions. The influences of AAIL concentration, pH, and methanol concentration on efficiency of chiral separation were investigated. Then it has been testified that the optimal buffer solution consisted of 25.0mM Cu(Ac)(2), 50.0 mM AAIL and 20% (v/v) methanol at pH 4.0. The interesting thing is well enantioresolution could be observed with [L-Pro][CF(3)COO] as the new chiral ligand and nine pairs of labeled D,L-AAs were successfully separated with the resolution ranging from 0.93 to 6.72. Meanwhile, the baseline separation of labeled D,L-AAs could be achieved with the other three kinds of AAILs as ligands. The results have demonstrated the good applicability of AAILs with AAs as cations for chiral separation in CLE-CE system. In addition, comparative study was also conducted for exploring the mechanism of the AAILs as new ligands in CLE-CE.

Chiral separation using capillary electromigration techniques based on ligand exchange principle.

Over the last couple of decades, researchers have developed diverse chiral separation methods emerged from a few chiral separation principles. This review article is primarily focused on the application of chiral ligand-exchange (CLE) principle in capillary electromigration techniques, such as capillary electrophoresis (CE) and capillary electrochromatography (CEC). First, the most commonly used CLE-CZE separation mode by using different kinds of central ions, such as Cu(II), Zn(II), borate ion, and other metal ions, has been introduced. Meanwhile, several kinds of surfactants have been applied as the micelle-forming agents in the CLE micellar electrokinetic chromatography mode. The highlight of recent research of CLE-CEC is the exploitation of novel columns for chiral separation. Then, two kinds of capillary columns, packed capillary and monolithic capillary column, have been briefly described. Finally, the effective application of these chiral separation methods has been presented, including the application in life science and food analysis area.

Study on the decrease of renal D-amino acid oxidase activity in the rat after renal ischemia by chiral ligand exchange capillary electrophoresis.

D: -Amino acid oxidase (DAAO) in mammal kidney regulates the renal reactive oxygen species (ROS) levels directly and plays a leading role in the development of ROS-mediated renal pathologic damages based on its crucial role in the oxidative deamination of D: -amino acids and the consequent generation of H(2)O(2). Quantitative measurement of DAAO activity in the process of renal ischemia, which could help to understand the molecular mechanisms of this gripping acute renal disease, was conducted through the determination of chiral substrate by capillary electrophoresis (CE) in our study. In this study, a chiral ligand exchange CE method was explored with Zn(II)-L: -alaninamide complex as the chiral selector to investigate DAAO activity by determining the decreased concentration of the chiral substrate of DAAO-mediated enzymatic reaction. Then, the change of DAAO activity following 60-min acute renal ischemia in rats was observed with the proposed method. The study showed that the operation of renal ischemia resulted in a 45.49 ± 8.30% (n = 8) decrease in the DAAO-induced consumption of substrate, indicating a sharp decrease in renal DAAO activity following this acute renal injury. This phenomenon, with the possible reason of metabolic acidosis, could pave a new way for the study of oxidative stress in the development of renal ischemia injury.

Study on alanine aminotransferase kinetics by microchip electrophoresis.

Alanine aminotransferase (ALT), which catalyzes the reversible conversion between L-glutamic acid (L-Glu) and L-alanine (L-Ala), is one of the most active aminotransferases in the clinical diagnosis of liver diseases. This work displays a microanalytical method for evaluating ALT enzyme kinetics using a microchip electrophoresis laser-induced fluorescence system. Four groups of amino acid (AA) mixtures, including the substrates of ALT (L-Glu and L-Ala), were effectively separated. Under the optimized conditions, the quantitative analysis of L-Glu and L-Ala was conducted and limits of detection (signal/noise=3) for L-Glu and L-Ala were 4.0 × 10⁻7 and 2.0 × 10⁻7 M, respectively. In the reaction catalyzed by ALT, enzyme kinetic constants were determined for both the forward and reverse reactions by monitoring the concentration decrease of substrate AAs (L-Ala and L-Glu), and the K(m) and V(max) values were 10.12 mM and 0.48 mM/min for forward reaction and 3.22 mM and 0.22 mM/min for reverse reaction, respectively. Furthermore, the applicability of this assay was assessed by analysis of real serum samples. The results demonstrated that the proposed method could be used for kinetic study of ALT and shows great potential in the real application.

Development of a new open-tubular capillary electrochromatography method for in vitro monitoring of toxic aromatic amines distribution in rat blood.

Exposure to aromatic amines from different industrial and agricultural activities entails a substantial risk of deleterious somatic effects, genetic damage and cancer development. Thus, a new and simple method for separation and analysis of aromatic amines has been developed by open-tubular capillary electrochromatography with a novel amphipathic block copolymer (poly(tert-butyl acrylate)(127)-block-poly(glycidyl methacrylate)(86)) coating based on its self-assembled property. Key factors affecting the separation efficiency of the test analytes, such as pH, buffer concentration and selective solvent, were studied in detail. Meanwhile, method validation was well evaluated by linearity (≥0.998), detection limit and recovery. Application of this developed protocol on in vitro monitoring of the target aromatic amines distribution in rat blood demonstrated its potential usage for separation and determination of aromatic amines in biological samples. Additionally, for assimilating more polymeric materials into analysis of aromatic amines, the effect of morphology changes of the amphipathic block copolymer coating on open-tubular capillary electrochromatography separation was also studied, and the result revealed that the block copolymer coating could play the same role as surfactant.

Determination of sodium benzoate by chiral ligand exchange CE based on its inhibitory activity in D-amino acid oxidase mediated oxidation of D-serine.

A novel quantitative approach for the determination of sodium benzoate (SB) was proposed by the kinetic study about its competitive inhibitory efficiency to D-amino acid oxidase (DAAO) activity with a chiral ligand exchange capillary electrophoresis (CE) method, in which the Zn(II)-L-prolinamide complex was chosen as a novel chiral selector. After the optimization of buffer pH and the chiral selector concentration this chiral ligand exchange CE method was employed to determine labeled D,L-Serine with good linearity (r(2)≥0.995), efficient recovery (95.6-100.9%) and remarkable reproducibility (RSD≤1.2%). This chiral separation method was further used to observe DAAO activity through the determination of D-Serine concentration variation after being incubated with DAAO and obtain the sigmoidal inhibitory curve of SB to DAAO activity. The ascending part of this inhibitory curve was linearly fitted in a limited range for SB from 2.0 to 200 µM with an appropriate coefficient of determination (R(2)=0.990). The linearity was then validated to be a promising method for the analysis of SB with the standout merits of high selectivity and adjustable detection range. Furthermore, this proposed method was used for the pharmacokinetics study of SB.