Highly electrochemically active Ti3C2Tx MXene/MWCNT nanocomposite for the simultaneous sensing of paracetamol, theophylline, and caffeine in human blood samples.

The facile fabrication is reported of highly electrochemically active Ti3C2Tx MXene/MWCNT (3D/1D)-modified screen-printed carbon electrode (SPE) for the efficient simultaneous electrochemical detection of paracetamol, theophylline, and caffeine in human blood samples. 3D/1D Ti3C2Tx MXene/MWCNT nanocomposite was synthesized using microwave irradiation and ultrasonication processes. Then, the Ti3C2Tx/MWCNT-modified SPE electrode was fabricated and thoroughly characterized towards its physicochemical and electrochemical properties using XPS, TEM, FESEM, XRD, electrochemical impedance spectroscopy, cyclic voltammetry, and differential pulse voltammetry techniques. As-constructed Ti3C2Tx-MWCNT/SPE offers excellent electrochemical sensing performance with good detection limits (0.23, 0.57, and 0.43 µM) and wide linear ranges (1.0 ~ 90.1, 2.0 ~ 62.0, and 2.0-90.9 µM) for paracetamol, caffeine, and theophylline, respectively,  in the human samples. Notably, the non-enzymatic electroactive nanocomposite-modified electrode has depicted a semicircle Nyquist plot with low charge transfer resistance (Rct∼95 Ω), leading to high ionic diffusion and facilitating an excellent electron transfer path. All the above results in efficient stability, reproducibility, repeatability, and sensitivity compared with other reported works, and thus, it claims its practical utilization in realistic clinical applications.

Lignin phenolation by graft copolymerization to boost its reactivity.

Lignin is the most abundant phenolic biopolymer and a renewable resource of aromatics. It can be used as a phenol substitute in the synthesis of phenolic resins. However, lignin is not as reactive as phenol, so phenolation is generally carried out to improve lignin reactivity. In this work, we suggest a solution to circumvent the limitations of traditional phenolation (e.g., high temperature, strong acids/bases, limited reactivity, and phenol toxicity). We first attempt new lignin phenolation by graft copolymerization in which polymeric phenol, instead of toxic phenol, is introduced to lignin. Organosolv lignin from hardwood was modified with 2-bromoisobutyryl bromide to act as a lignin macroinitiator (L-Br). A protected phenolic monomer, 4-acetoxystyrene, was graft copolymerized onto L-Br using CuBr2/tris[2-(dimethylamino)ethyl]amine as a catalyst/ligand, after which the resultant lignin copolymer was deacetylated to produce lignin grafted with poly(4-hydroxystyrene). This poly-phenolation process was conducted at room temperature without the strong acids/bases and toxic phenol required in conventional phenolation. The poly-phenolated lignin was analyzed using 1H-, 13C-, and 31P NMR spectroscopy and gel permeation chromatography. This novel phenolation process enhanced the reactive sites of lignin more than tenfold. It also reduced the dark color of technical lignins significantly, thereby overcoming a serious obstacle to their applicability.

Solvent-free enzymatic synthesis and evaluation of vanillyl propionate as an effective and biocompatible preservative.

Preservatives are chemicals added to protect products against microbial spoilage, and thus are indispensable for pharmaceuticals, cosmetics, and foods. Due to growing concerns about human health and environments in conventional chemical preservatives, many companies have been seeking safe and effective alternatives that can be produced through environment-friendly processes. In this work, in order to develop effective and safe preservatives from plants, we attempt solvent-free lipase-catalyzed transesterification of vanillyl alcohol with ethyl propionate for the first time. The reaction product, vanillyl propionate was efficiently obtained in a high yield. Unlike vanillyl alcohol and ethyl propionate, vanillyl propionate showed antimicrobial activity. The minimal inhibitory concentration test showed that it exhibited high and broad antimicrobial activity against all the tested microorganisms (Gram-negative and Gram-positive bacteria, yeasts, and molds), which was overall comparable to that of propyl paraben, which is one of the most effective preservatives. It was also found to have even higher antioxidant capacity and biocompatibility with human cells than propyl paraben. Vanillyl propionate, which is a plant-based preservative produced through a green bioprocess, is expected to be successfully applied to various industries thanks to its high antimicrobial and antioxidant effect, and high biocompatibility.

Preparation and Application of Light-Colored Lignin Nanoparticles for Broad-Spectrum Sunscreens.

Recently, natural sun blockers have been drawing considerable attention because synthetic UV filters could have adverse effects not only on humans but also on the environment. Even though lignin, the second most abundant renewable resource on earth, is a natural UV-absorbing polymer, its unfavorable dark color hampers its applications in sunscreens. In this work, we obtained light-colored lignin (CEL) from rice husks through cellulolytic enzyme treatment and subsequent solvent extraction under mild conditions and compared CEL to technical lignin from rice husks using the International Commission on Illumination L*a*b* (CIELAB) color space. Spherical nanoparticles of CEL (CEL-NP) were also prepared using a solvent shifting method and evaluated for broad-spectrum sunscreens. A moisturizing cream blended with CEL-NP exhibited higher sun protection factor (SPF) and UVA PF (protection factor) values than that with CEL. In addition, CEL-NP had synergistic effects when blended with an organic UV-filter sunscreen: CEL-NP enhanced the SPF and UVA PF values of the sunscreen greatly. However, there was no synergistic effect between CEL-NP and inorganic sunscreens. We expect nanoparticles of light-colored lignin to find high-value-added applications as a natural UV-blocking additive in sunscreens and cosmetics.

Lignin for white natural sunscreens.

Long-time exposure to the sun's ultraviolet (UV) radiation is harmful and causes various skin problems. Natural sun blockers have been drawing considerable attention recently. Even though lignin, an abundant aromatic polymer from plants, is a natural UV screening agent, its unfavorable dark color hinders its high value-added applications in sunscreens and cosmetics. In this study, we separate lignin under mild conditions (at room temperature with neutral solvents) in order to prevent darkening occurring during delignification and apply the resultant lignin as a natural sunscreen ingredient for the first time. Lignins isolated from Miscanthus sacchariflorus (MWL-M) and from Pinus densiflora (MWL-P) are compared with organosolv lignin (OL), which showed the best sunscreen performance, in color and UV protection. MWLs separated under mild conditions were light in color unlike conventional lignins extracted under harsh conditions. UV absorption of light-colored MWL-M was revealed to be as high as dark-colored OL. MWLs also showed synergistic effects with a commercial sunscreen; exposure of the MWL-added sunscreen to UVA radiation greatly enhanced the sun protection factor (SPF) value of the sunscreen.

Preparation of poly(lactide)/lignin/silver nanoparticles composite films with UV light barrier and antibacterial properties.

Organosolv lignin was used as a reducing agent for the preparation of silver nanoparticles (AgNPs) and their incorporation into poly(lactide) (PLA) polymer to prepare composite films. The composite films were characterized using UV-vis spectroscopy, FE-SEM, FTIR, XRD, and TGA. The optical, mechanical, water vapor barrier, and antibacterial properties of the composite films were evaluated. The UV-vis spectra of films exhibited two characteristics peaks around 300 and 450nm attributed to lignin and AgNPs, respectively. XRD results indicated that the crystalline AgNPs had been formed. The transmission of light at 280nm decreased significantly after incorporation of lignin and AgNPs. FTIR results showed that there was no change in the chemical structure of PLA after incorporation of lignin and AgNPs. The mechanical and water vapor barrier properties of the composite films increased after lignin and AgNPs incorporation, The films containing AgNPs exhibited potent antibacterial activity against Escherichia coli and Listeria monocytogenes.

A Natural Component-Based Oxygen Indicator with In-Pack Activation for Intelligent Food Packaging.

Intelligent food packaging can provide consumers with reliable and correct information on the quality and safety of packaged foods. One of the key constituents in intelligent packaging is a colorimetric oxygen indicator, which is widely used to detect oxygen gas involved in food spoilage by means of a color change. Traditional oxygen indicators consisting of redox dyes and strong reducing agents have two major problems: they must be manufactured and stored under anaerobic conditions because air depletes the reductant, and their components are synthetic and toxic. To address both of these serious problems, we have developed a natural component-based oxygen indicator characterized by in-pack activation. The conventional oxygen indicator composed of synthetic and artificial components was redesigned using naturally occurring compounds (laccase, guaiacol, and cysteine). These natural components were physically separated into two compartments by a fragile barrier. Only when the barrier was broken were all of the components mixed and the function as an oxygen indicator was begun (i.e., in-pack activation). Depending on the component concentrations, the natural component-based oxygen indicator exhibited different response times and color differences. The rate of the color change was proportional to the oxygen concentration. This novel colorimetric oxygen indicator will contribute greatly to intelligent packaging for healthier and safer foods.

Wood mimetic hydrogel beads for enzyme immobilization.

Wood component-based composite hydrogels have potential applications in biomedical fields owing to their low cost, biodegradability, and biocompatibility. The controllable properties of wood mimetic composites containing three major wood components are useful for enzyme immobilization. Here, lipase from Candida rugosa was entrapped in wood mimetic beads containing cellulose, xylan, and lignin by dissolving wood components with lipase in [Emim][Ac], followed by reconstitution. Lipase entrapped in cellulose/xylan/lignin beads in a 5:3:2 ratio showed the highest activity; this ratio is very similar to that in natural wood. The lipase entrapped in various wood mimetic beads showed increased thermal and pH stability. The half-life times of lipase entrapped in cellulose/alkali lignin hydrogel were 31- and 82-times higher than those of free lipase during incubation under denaturing conditions of high temperature and low pH, respectively. Owing to their biocompatibility, biodegradability, and controllable properties, wood mimetic hydrogel beads can be used to immobilize various enzymes for applications in the biomedical, bioelectronic, and biocatalytic fields.

Leaching-resistant carrageenan-based colorimetric oxygen indicator films for intelligent food packaging.

Visual oxygen indicators can give information on the quality and safety of packaged food in an economic and simple manner by changing color based on the amount of oxygen in the packaging, which is related to food spoilage. In particular, ultraviolet (UV)-activated oxygen indicators have the advantages of in-pack activation and irreversibility; however, these dye-based oxygen indicator films suffer from dye leaching upon contact with water. In this work, we introduce carrageenans, which are natural sulfated polysaccharides, to develop UV-activated colorimetric oxygen indicator films that are resistant to dye leakage. Carrageenan-based indicator films were fabricated using redox dyes [methylene blue (MB), azure A, and thionine], a sacrificial electron donor (glycerol), an UV-absorbing photocatalyst (TiO2), and an encapsulation polymer (carrageenan). They showed even lower dye leakage in water than conventional oxygen indicator films, owing to the electrostatic interaction of anionic carrageenan with cationic dyes. The MB/TiO2/glycerol/carrageenan oxygen indicator film was successfully bleached upon UV irradiation, and it regained color very rapidly in the presence of oxygen compared to the other waterproof oxygen indicator films.

Novel water-resistant UV-activated oxygen indicator for intelligent food packaging.

For the first time, alginate polymer has been applied to prevent dyes from leaching out of colorimetric oxygen indicator films, which enable people to notice the presence of oxygen in the package in an economic and simple manner. The dye-based oxygen indicator film suffers from dye leaching upon contact with water. In this work, UV-activated visual oxygen indicator films were fabricated using thionine, glycerol, P25 TiO2, and zein as a redox dye, a sacrificial electron donor, UV-absorbing semiconducting photocatalyst, and an encapsulation polymer, respectively. When this zein-coated film was immersed in water for 24h, the dye leakage was as high as 80.80±0.45%. However, introduction of alginate (1.25%) as the coating polymer considerably diminished the dye leaching to only 5.80±0.06%. This is because the ion-binding ability of alginate could prevent the cation dye from leaching into water. This novel water-resistant UV-activated oxygen indicator was also successfully photo-bleached and regained colour fast in the presence of oxygen.

Glucose oxidase/cellulose-carbon nanotube composite paper as a biocompatible bioelectrode for biofuel cells.

Biofuel cells are devices for generating electrical energy directly from chemical energy of renewable biomass using biocatalysts such as enzymes. Efficient electrical communication between redox enzymes and electrodes is essential for enzymatic biofuel cells. Carbon nanotubes (CNTs) have been recognized as ideal electrode materials because of their high electrical conductivity, large surface area, and inertness. Electrodes consisting entirely of CNTs, which are known as CNT paper, have high surface areas but are typically weak in mechanical strength. In this study, cellulose (CL)-CNT composite paper was fabricated as electrodes for enzymatic biofuel cells. This composite electrode was prepared by vacuum filtration of CNTs followed by reconstitution of cellulose dissolved in ionic liquid, 1-ethyl-3-methylimidazolium acetate. Glucose oxidase (GOx), which is a redox enzyme capable of oxidizing glucose as a renewable fuel using oxygen, was immobilized on the CL-CNT composite paper. Cyclic voltammograms revealed that the GOx/CL-CNT paper electrode showed a pair of well-defined peaks, which agreed well with that of FAD/FADH2, the redox center of GOx. This result clearly shows that the direct electron transfer (DET) between the GOx and the composite electrode was achieved. However, this DET was dependent on the type of CNTs. It was also found that the GOx immobilized on the composite electrode retained catalytic activity for the oxidation of glucose.

Silaffin peptides as a novel signal enhancer for gravimetric biosensors.

Application of biomimetic silica formation to gravimetric biosensors has been conducted for the first time. As a model system, silaffin peptides fused with green fluorescent protein (GFP) were immobilized on a gold quartz crystal resonator for quartz crystal microbalances using a self-assembled monolayer. When a solution of silicic acid was supplied, silica particles were successfully deposited on the Au surface, resulting in a significant change in resonance frequency (i.e., signal enhancement) with the silaffin-GFP. However, frequency was not altered when bare GFP was used as a control. The novel peptide enhancer is advantageous because it can be readily and quantitatively conjugated with sensing proteins using recombinant DNA technology. As a proof of concept, this study shows that the silaffin domains can be employed as a novel and efficient biomolecular signal enhancer for gravimetric biosensors.

Bioinspired molecular adhesive for water-resistant oxygen indicator films.

Mussels can attach themselves to nearly all types of hard surfaces in wet environments. Such attractive adhesive ability of mussels is believed to rely on the amino acid composition of proteins found near the plaque-substrate interface. Dopamine (DA) is identified as a simplified mimic of mussel proteins, which are rich in 3,4-dihydroxy-L-phenylalanine and lysine, because it contains both catechol and amine functional groups. In this work, we have first applied this bioinspired adhesive to tackle a dye leaching problem of colorimetric oxygen indicator films, which are widely used to ensure the absence of oxygen inside the package of oxygen-sensitive materials. Simple immersion of packaging films into a DA solution resulted in poly(DA) deposition, decreasing the water contact angle of the films from 105° to 65°. The poly(DA) coating could reduce the thionine leakage of the UV-activated oxygen indicator film. The effects of poly(DA) coating were found to be dependent on the DA solution pH, the coating time, and the DA concentration. The film resistant to dye leaching lost its dye color by 5 min UVB irradiation and regained the color in the presence of oxygen, demonstrating that it functioned successfully as UV-activated oxygen indicators.

In vitro analysis of the monolignol coupling mechanism using dehydrogenative polymerization in the presence of peroxidases and controlled feeding ratios of coniferyl and sinapyl alcohol.

In this study, dehydrogenative polymers (DHP) were synthesized in vitro through dehydrogenative polymerization using different ratios of coniferyl alcohol (CA) and sinapyl alcohol (SA) (10:0, 8:2, 6:4, 2:8, 0:10), in order to investigate the monolignol coupling mechanism in the presence of horseradish peroxidase (HRP), Coprinus cinereus peroxidase (CiP) or soybean peroxidase (SBP) with H(2)O(2), respectively. The turnover capacities of HRP, CiP and SBP were also measured for coniferyl alcohol (CA) and sinapyl alcohol (SA), and CiP and SBP were found to have the highest turnover capacity for CA and SA, respectively. The yields of HRP-catalyzed DHP (DHP-H) and CiP-catalyzed DHP (DHP-C) were estimated between ca. 7% and 72% based on the original weights of CA/SA in these synthetic conditions. However, a much lower yield of SBP-catalyzed DHP (DHP-S) was produced compared to that of DHP-H and DHP-C. In general, the DHP yields gradually increased as the ratio of CA/SA increased. The average molecular weight of DHP-H also increased with increasing CA/SA ratios, while those of DHP-C and DHP-S were not influenced by the ratios of monolignols. The frequency of ß-O-4 linkages in the DHPs decreased with increasing CA/SA ratios, indicating that the formation of ß-O-4 linkages during DHP synthesis was influenced by peroxidase type.

Artificial electron carriers for photoenzymatic synthesis under visible light.

NAD analogues can be employed as artificial electron carriers for photoenzymatic synthesis under visible light. Four different NAD analogues that have a 3-substituted pyridine ring have been investigated. 3-Acetylpyridine adenine dinucleotide and 3-pyridinealdehyde adenine dinucleotide were photochemically reduced much more efficiently than NAD, while their reduced products showed coenzyme activity comparable to natural NAD.

Coenzyme analogs: excellent substitutes (not poor imitations) for electrochemical regeneration.

For the first time, employment of nicotinamide coenzyme NAD analogs has overcome the limitations of NAD in electrochemical regeneration. It has been shown that NAD analogs, APAD and PAAD, were electrochemically reduced more efficiently than original NAD and that the stability of their reduced products was also much higher than NADH.

Structural features of lignin macromolecules extracted with ionic liquid from poplar wood.

1-Ethyl-3-methylimidazolium acetate ([Emim][CH3COO]) was used for the extraction of lignin from poplar wood (Populus albaglandulosa), which was called to ionic liquid lignin (ILL) and structural features of ILL were compared with the corresponding milled wood lignin (MWL). Yields of ILL and MWL were 5.8±0.3% and 4.4±0.4%, respectively. The maximum decomposition rate (V(M)) and temperature (T(M)) corresponding to V(M) were 0.25%/ °C and 308.2 °C for ILL and 0.30%/ °C and 381.3 °C for MWL. The amounts of functional groups (OMe and phenolic OH) appeared to be similar for both lignins; approximately 15.5% and 6.7% for ILL and 14.4% and 6.3% for MWL. However, the weight average molecular weight (M(w)) of ILL (6347 Da) was determined to be 2/3-fold of that of MWL (10,002 Da) and polydispersity index (PDI: M(w)/M(n)) suggested that the lignin fragments were more uniform in the ILL (PDI 1.62) than in the MWL (PDI 2.64).

A novel route for immobilization of proteins to silica particles incorporating silaffin domains.

In the diatom Cylindrotheca fusiformis, modified peptides called silaffin polypeptides are responsible for silica deposition in vivo at ambient conditions. Recently, it was discovered that the synthetic R5 peptide, the repeat unit of silaffin polypeptide without post-translational modification, was capable of precipitating silica in vitro and at ambient conditions. Herein, chimeric proteins were generated by incorporating synthetic silaffin R5 peptides and related unmodified silaffin domains (R1-R7) from Cylindrotheca fusiformis onto green fluorescent protein (GFP) by recombinant DNA technology and their ability to cause silicification was also examined. GFP chimeric proteins showed silicification at very low concentrations (600-700 microg/mL) when compared with adding excess amounts of R5 peptides (10 mg/mL) as previously reported. Sensitive to pH conditions, only the GFP-R1 chimera showed silicification activity at pH 8.0. The protein immobilization efficiencies of these chimeras were unexpectedly high ranging from 75 to 85%, with the R1 silaffin-protein construct showing excellent immobilization efficiency and a constant molar ratio of silica to protein ranging from 250 to 350 over a wide pH range. The average silica particle sizes had a tendency to decrease as pH increased to basic conditions. This study demonstrated the production of nanoscale immobilized protein, fabricated via silaffin-fused proteins.

Immobilization and characterization of ferritin on gold electrode.

Properties of ferritin, immobilized on dithiobis (N-succinimydyl propionate) (DTSP)-covered gold electrode, in 3-morpholino propanesulfonic acid buffer were investigated by AFM and FE-SEM. Electrochemical properties the ferritin was measured by a cyclic volatammetry. When the potentials of 0.2, 0.34, and 1.0V vs. Ag/AgCl were applied for 15h for the ferritin immobilization, the electrode potential of Fe(II)/Fe(III) in ferritin changed to negative values. Negative electrode potential shift of Fe(II)/Fe(III) in ferritin with respect to the applied potential could be attributed to the stabilized ferritin on DTSP-covered gold electrode. From AFM and SEM images, it was proven that ferritins fixed at below 0.34V were clusters in several micrometer and those fixed at higher applied potential than OCV were finely distributed particles in several tens of nanometer.