Semiquinone Radical Research Articles

Electron Paramagnetic Resonance Analysis of Hydroquinone Polymerization Catalyzed by Small Laccase

AbstractLaccases are multi‐copper oxidases (MCOs) that oxidize a broad range of substrates while reducing molecular oxygen to water. Although much effort has been made to elucidate the catalytic mechanism of laccases, information about reactive catalytic intermediates during catalysis is not yet fully understood. Herein, hydroquinone (HQ) polymerization catalyzed by prokaryotic small laccase (SLAC) was investigated by electron paramagnetic resonance (EPR) spectroscopy to provide more information on the catalytic mechanism. Briefly, free radical intermediates during the catalysis were investigated by real‐time in situ EPR measurement to monitor the formation and transformation of free radicals. A paramagnetic species was identified which was attributed to a copolymer formed by hydroquinone, benzoquinone, and semiquinone radical. In addition, the low‐temperature EPR spectrum of the trinuclear copper center during catalysis not only revealed tentative rotational motion of the histidine imidazole rings coordinating the TNC upon electron uptake but also provided evidence of the formation of oxygen bridge at TNC during the reaction, represented by the observation of a new type 2 copper component featured larger g// values and smaller A// values. Together, EPR spectroscopic insights into the catalytic intermediates during enzymatic hydroquinone polymerization have extended the knowledge of the biophysical characteristics and catalytic mechanism of SLAC.

Analyzing the FMN-heme interdomain docking interactions in neuronal and inducible NOS isoforms by pulsed EPR experiments and conformational distribution modeling.

Nitric oxide synthases (NOSs), a family offlavo-hemoproteins with relatively rigid domains linked by flexible regions, require optimal FMN domain docking to the heme domain for efficient interdomain electron transfer (IET). To probe the FMN-heme interdomain docking, the magnetic dipole interactions between the FMN semiquinone radical (FMNH•) and the low-spin ferric heme centers in oxygenase/FMN (oxyFMN) constructs of neuronal and inducible NOS (nNOS and iNOS, respectively) were measured using the relaxation-induced dipolar modulation enhancement (RIDME) technique. The FMNH•RIDME data were analyzed using the mesoscale Monte Carlo calculations of conformational distributions of NOS, which were improved to account for the native degrees of freedom of the amino acid residues constituting the flexible interdomain tethers. This combined computational and experimentalanalysis allowed for the estimation of the stabilization energies and populations of the docking complexes of calmodulin (CaM) and the FMN domain with the heme domain. Moreover, combining the five-pulse and scaled four-pulse RIDME data into a single trace has significantly reduced the uncertainty in the estimated docking probabilities. The obtained FMN-heme domain docking energies for nNOS and iNOS were similar (-3.8kcal/mol), in agreement with the high degree of conservation of the FMN-heme domain docking interface between the NOS isoforms. In spite of the similar energetics, the FMN-heme domain docking probabilities in nNOS and iNOS oxyFMN were noticeably different (~ 0.19 and 0.23, respectively), likely due to differences in the lengths of the FMN-heme interdomain tethers and the docking interface topographies. The analysis based on the IET theory and RIDME experiments indicates that the variations in conformational dynamics may account for half of the difference in the FMN-heme IET rates between the two NOS isoforms.

Carbon Nanosheet Preparation By Low-Temperature Two-Step Carbonization: A Study of Their Properties and Mechanism of Adsorption of Oxidized H2S.

Straw, as a kind of biomass waste, has the advantages of low cost and abundant storage, which makes it a promising renewable resource. Using rice straw as a carbon source, carbon nanosheets were prepared by a two-step carbonization method combining low-temperature pyrolysis and low-temperature hydrothermal, and they were used as H2S removal agents. The results showed that during the two-step carbonization process, the adsorption performance of carbon nanosheets for H2S showed a tendency of enhancing and then weakening with the increase of pyrolysis temperature in the first step, and the sulfur capacity could reach 3.1 mg/g at the maximum of the pyrolysis temperature of 200 °C, which was superior to or close to that of the modified or activated carbon. The XPS, EPR, and CO2-TPD tests showed that the surface of carbon nanosheets was alkaline, containing a large number of hydroxyl groups and the presence of phenoxy persistent free radicals or semiquinone persistent free radicals. It was analyzed that the direct or indirect oxidation of H2S by the persistent radicals under an alkaline environment could convert the -2-valent sulfur into -1-, 0- and +6-valent sulfur to realize the adsorption and removal of H2S. This work, while offering the possibility of utilizing carbon nanosheets made from straw as a material for H2S adsorption and removal, also expands the application of straw waste in exhaust gas treatment.

Electrochemistry of Diquinonyl Amines with an Internal Proton Source

It is well established that quinones play an important role in various biological systems. Studying electrochemical properties of quinones affords fundamental knowledge of semi-quinone radicals formation in vivo and in vitro in different media.Following our previous work on electrochemical properties of various quinonyl amines [1], Scheme 1 below describes seven diquinonyl amines. Noteworthy that six of them (1-6) contain an internal proton donor (‘NH’) except for the seventh one (7), in which both quinone moieties are attached to ‘NMe’ group. Their redox potentials were measured by cyclic voltammetry in dichloromethane [2].The results show a strong dependence of the nature of substituent on the first reduction potentials, and that protonation of diquinonyl amines is feasible by internal proton source even in a non-polar medium.

Nature-Inspired Radical Pyridoxal-Mediated C-C Bond Formation.

Pyridoxal-5'-phosphate (PLP) and derivatives of this cofactor enable a plethora of reactions in both enzyme-mediated and free-in-solution transformations. With few exceptions in each category, such chemistry has predominantly involved two-electron processes. This sometimes poses a significant challenge for using PLP to build tetrasubstituted carbon centers, especially when the reaction is reversible. The ability to access radical pathways is paramount to broadening the scope of reactions catalyzed by this coenzyme. In this study, we demonstrate the ability to access a radical PLP-based intermediate and engage this radical intermediate in a number of C-C bond-forming reactions. By selection of an appropriate oxidant, single-electron oxidation of the quinonoid intermediate can be achieved, which can subsequently be applied to C-C bond-forming reactions. Through this radical reaction pathway, we synthesized a series of α-tertiary amino acids and esters to investigate the substrate scope and identify nonproductive reaction pathways. Beyond the amino acid model system, we demonstrate that other classes of amine substrates can be applied in this reaction and that a range of small molecule reagents can serve as coupling partners to the semiquinone radical. We anticipate that this versatile semiquinone radical species will be central to the development of a range of novel reactions.

Fluorescence detection of valence speciation of Cr(III) based on the catechol oxidase mimic enzyme activity of CuSeNP nanozymes.

Copper selenide nanoparticles (CuSeNP) weresynthesized using histidine, ethylenediamine, and sodium selenate as precursors by one-step microwave digestion methods. The as-prepared CuSeNPs exhibit excellent catechol oxidase mimic enzyme and catalase (CAT)-like activities. Dopamine (DA) can be oxidized to aminochrome with H2O2 by CuSeNPs, and the intermediate product aminochrome can further react with α-naphthol to yield a highly fluorescent derivative. It was confirmed that Cr(III) could adsorb on the surface of CuSeNPs and inhibit the production of semiquinone radicals in the reaction system, and the catalytic activity of CuSeNPs was inhibited. The detection mechanisms, kinetics, and catalytic properties of CuSeNPs were systematically investigated. As a result, a novel fluorescence method for the assay of Cr(III) was established. The feasibility of CuSeNP nanozyme in detecting speciation Cr(III) in food samples was explored with satisfactory results. It showed the obvious potential for developing effective and dependable fluorescent detection method for protecting food safety.

Importance of Polarizable Embedding for Absorption Spectrum Calculations of Arabidopsis thaliana Cryptochrome 1.

Cryptochromes are essential flavoproteins for circadian rhythms and avian magnetoreception. Flavin adenine dinucleotide (FAD), a chromophore within cryptochromes, absorbs blue light, initiating electron transfer processes that lead to a biological signaling cascade. A key step in this cascade is the formation of the FAD semiquinone radical (FADH•), characterized through a specific red-light absorption. The absorption spectra of FADH• in cryptochromes are, however, significantly different from those recorded for the cofactor in solution, primarily due to protein-induced shifts in the absorption peaks. This study employs a multiscale approach, combining molecular dynamics (MD) simulations with quantum mechanical/molecular mechanical (QM/MM) methodologies, to investigate the influence of protein dynamics on embedded FADH• absorption. We emphasize the role of the protein's polarizable environment in the shaping of the absorption spectrum, crucial for accurate spectral predictions in cryptochromes. Our findings provide valuable insights into the absorption process, advancing our understanding of cryptochrome functioning.

Impact of biochar synergized bacteria on Mn2+ and NH4+-N removal and CO2 immobilization in water by enhanced extracellular electron transfer

Mn2+ and NH4+-N contamination in water by Mn slag leakage is urgent to be resolved. Biochar synergistic strains is considered to be a promising approach to remove pollution with high concentration, however, the specific oxidation and electron transfer processes are rarely elucidated in detail. In this study, under different biochar (BC) additions, NH4+-N concentration and pH ranges, removal efficiency of Mn2+, NH4+-N and COD had reached at 99.7 %-100 %, 100 % and 90.3–96.2 % by the BC & strain AL-6 system (co-system), respectively, and the nitrification processes was completed. Meanwhile, the electrochemical analysis showed that BC had strong tendency to donate, accept and transfer electrons, and electrons transfer property was more responsible for the removal process. Further, the electrical conductivity of co-system was enhanced upon reaction with Mn2+ and NH4+-N, suggesting that co-system promoted the electron transfer processes for pollutants removal. Concretely, Raman spectroscopy specified that conductive graphite region of biochar accelerated electron transfer behavior in the system. As well as, 2D-COS and characterization analyses indicated that C-C and CC functional groups, O2•− radicals, and semiquinone radicals could promote the oxidation of manganese. Eventually, manganese presented 2+ and high valence state was adsorbed on the surface of the co-system. In addition, BC facilitated strain AL-6 to convert organic carbon into CO2 and transfer electrons for N metabolism, and over time, CO2 and N2O could also be absorbed by BC. This study further dissected the efficiency on the Mn-contaminated water bodies and mechanism process by co-system and provided potential value in mitigating climate change.

Catalysis of first triplet p-benzoquinone for electron ejection from sulfite: Significance of single electron transfer

In this study, the combination of p-benzoquinone (p-BQ) and sulfite under the ultraviolet (UV) irradiation demonstrated markedly superior efficiency in the ejection of hydrated electrons (eaq−) compared to the individual UV/p-BQ and UV/sulfite processes. Theoretical calculations reveal that the 1,2- and 1,4-addition of bisulfite (HSO3−) to p-BQ was unlikely to occur kinetically, and the 1,4-addition of sulfite (SO32−) might be the major pathway for the reaction with p-BQ. Via single electron transfer from the highest occupied molecular orbitals (HOMOs) of HSO3− and SO32− to the HOMO–1 of 3p-BQ*, the oxidation of HSO3− and SO32− by the first triplet state of p-BQ (3p-BQ*, the major excited species during the excitation) could result in the generation of HSO3•, SO3•− and semiquinone radical anion. This mechanism could elucidate the synergism of p-BQ and sulfite for ejecting eaq− in the water under the UV irradiation. Given the abundance of quinones in natural waters, these findings may be helpful for the utilization of sulfite to efficiently release eaq− in the water treatment applications.

Coral-like silver citrate as robust laccase mimetics in a novel colorimetric aptasensor for sensitive and highly selective detection of histamine in food

The colorimetric methods for histamine detection still face some challenges in accuracy and stability. Herein, a novel colorimetric aptasensor for sensitive and highly selective detection of histamine was developed based on the enhancement of silver citrate (AgCit) laccase-mimic activity. It has been confirmed that H2 aptamer enhances the affinity of AgCit to the chromogenic substrate, and promotes the generation of semiquinone radical in the laccase-mimic catalytical system. The color change of the sensing solution can be distinguished by the naked eye, and easily converted to the corresponding RGB value by a smartphone for the quantitative and rapid detection of histamine. The aptasensor has a limit of detection (LOD) as low as 27 μg L−1, and maintains high selectivity for histamine in the presence of other competitive substances. It is strongly believed that the design of a colorimetric aptasensor can provide a simple and versatile platform for histamine detection in food.

Binding and Stabilization of a Semiquinone Radical by an Artificial Metalloenzyme Containing a Binuclear Copper (II) Cofactor.

A binuclear Cu(II) cofactor was covalently bound to a lauric acid anchor. The resulting conjugate was characterized then combined with beta-lactoglobulin (βLG) to generate a new biohybrid following the so-called "Trojan horse" strategy. This biohybrid was examined for its effectiveness in the oxidation of a catechol derivative to the corresponding quinone. The resulting biohybrid did not exhibit the sought after catecholase activity, likely due to its ability to bind and stabilize the semiquinone radical intermediate DTB-SQ. This semi-quinone radical was stabilized only in the presence of the protein and was characterized using optical and magnetic spectroscopic techniques, demonstrating stability for over 16 hours. Molecular docking studies revealed that this stabilization could occur owing to interactions of the semi-quinone with hydrophobic amino acid residues of βLG.

Neglected sludge solid phase in sludge pretreatment process: Physicochemical characterization and mechanism study of its role in anaerobic degradation

The low anaerobic digestion efficiency of the solid phase separated from pre-treated sludge indicates the need to explore other suitable resource utilization pathways for sludge solid phase. However, there is a lack of comprehensive and in-depth research on the physicochemical properties of sludge solid phase. This study comprehensively analyzes the characteristics of sludge solid phase and elucidates the mechanism of sludge solid phase in the anaerobic degradation of toxic wastewater. The results show that the surface free energy of sludge solid phase after different pre-treatments is mainly contributed by Lewis acid-base hydration free energy. The distribution of proteins on the surface of sludge solid phase plays a major role in the adhesion between sludge solid particles. Metal ions in the sludge solid phase are mainly present in the exchange state, followed by the carbonate state and the organics-bound state. The sludge solid phase obtained by sludge pH 12 + 150 °C treatment has the highest conductivity (1.36 mS/m) and capacitance (25.51 μF/g), mainly due to the presence of melanoidins in the sludge solid phase, which has similar semiquinone radicals to humic acids, thus increasing conductivity. The addition of sludge solid phase promotes an increase in cumulative methane production and rate of methane production. The sludge solid phase might play a role of an auxiliary carbon source acting as an adsorbent to buffer against toxicity inhibition and facilitate electron transfer. This study reveals the characteristics of sludge solid phase and its role in anaerobic digestion, providing theoretical guidance for finding suitable resource utilization pathways for sludge solid phase.

Ultrafast Spectroelectrochemistry of the Catechol/o‐Quinone Redox Couple in Aqueous Buffer Solution

AbstractEumelanin is a natural pigment found in many organisms that provides photoprotection from harmful UV radiation. As a redox‐active biopolymer, the structure of eumelanin is thought to contain different redox states of quinone, including catechol subunits. To further explore the excited state properties of eumelanin, we have investigated the catechol/o‐quinone redox couple by spectroelectrochemical means, in a pH 7.4 aqueous buffered solution, and using a boron doped diamond mesh electrode. At pH 7.4, the two proton, two electron oxidation of catechol is promoted, which facilitates continuous formation of the unstable o‐quinone product in solution. Ultrafast transient absorption (femtosecond to nanosecond) measurements of o‐quinone species involve initial formation of an excited singlet state followed by triplet state formation within 24 ps. In contrast, catechol in aqueous buffer leads to formation of the semiquinone radical Δt>500 ps. Our results demonstrate the rich photochemistry of the catechol/o‐quinone redox couple and provides further insight into the excited state processes of these key building blocks of eumelanin.

Metal-Organic Framework-Based Nanovaccine for Relieving Immunosuppressive Tumors via Hindering Efferocytosis of Macrophages and Promoting Pyroptosis and Cuproptosis of Cancer Cells.

Current cancer vaccines face challenges due to an immunosuppressive tumor microenvironment and their limited ability to produce an effective immune response. To address the above limitations, we develop a 3-(2-spiroadamantyl)-4-methoxy-4-(3-phosphoryloxy)-phenyl-1,2-dioxetane (alkaline phosphatase substrate) and XMD8-92 (extracellular signal-regulated kinase 5 inhibitor)-codelivered copper-tetrahydroxybenzoquinone (Cu-THBQ/AX) nanosized metal-organic framework to in situ-generate therapeutic vaccination. Once inside the early endosome, the alkaline phosphatase overexpressed in the tumor cells' membrane activates the in situ type I photodynamic effect of Cu-THBQ/AX for generating •O2-, and the Cu-THBQ/AX catalyzes O2 and H2O2 to •O2- and •OH via semiquinone radical catalysis and Fenton-like reactions. This surge of ROS in early endosomes triggers caspase-3-mediated proinflammatory pyroptosis via activating phospholipase C. Meanwhile, Cu-THBQ/AX can also induce the oligomerization of dihydrolipoamide S-acetyltransferase to trigger tumor cell cuproptosis. The production of •OH could also trigger the release of XMD8-92 for effectively inhibiting the efferocytosis of macrophages to convert immunosuppressive apoptosis of cancer cells into proinflammatory secondary necrosis. The simultaneous induction of pyroptosis, cuproptosis, and secondary necrosis effectively converts the tumor microenvironment from "cold" to "hot" conditions, making it an effective antigen pool. This transformation successfully activates the antitumor immune response, inhibiting tumor growth and metastasis.

Red Shift in the Absorption Spectrum of Phototropin LOV1 upon the Formation of a Semiquinone Radical: Reconstructing the Orbital Architecture.

Flavin mononucleotide (FMN) is a ubiquitous blue-light pigment due to its ability to drive one- and two-electron transfer reactions. In both light-oxygen-voltage (LOV) domains of phototropin from the green algae Chlamydomonas reinhardtii, FMN is noncovalently bound. In the LOV1 cysteine-to-serine mutant (C57S), light-induced electron transfer from a nearby tryptophan occurs, and a transient spin-correlated radical pair (SCRP) is formed. Within this photocycle, nuclear hyperpolarization is created by the solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect. In a side reaction, a stable protonated semiquinone radical (FMNH·) forms undergoing a significant bathochromic shift of the first electronic transition from 445 to 591 nm. The incorporation of phototropin LOV1-C57S into an amorphous trehalose matrix, stabilizing the radical, allows for application of various magnetic resonance experiments at ambient temperatures, which are combined with quantum-chemical calculations. As a result, the bathochromic shift of the first absorption band is explained by lifting the degeneracy of the molecular orbital energy levels for electrons with alpha and beta spins in FMNH· due to the additional electron.

INTERACTION OF (4E)-3-ALKYL(BENZYL)AMINO-4-(HYDROXYIMINO)NAPHTHALENE-1(4H)-ONES WITH 2,2-DIHYDROXY-1,3-INDANEDIONE

Many natural and synthetic quinone, including naphthoquinone derivatives, have some valuable properties. They are known as dyes, antibiotics, vitamins, and antioxidants. Some naphthoquinone derivatives act as cytostatic and antitumor reagents. However, the reduction of the quinoid fragment to the semiquinone radical and, as a consequence, the formation of toxic anion radicals, makes these substances cardiotoxic. The replacement of one or both carbonyl groups with the imino fragment can be one of the ways to solve this problem because iminoquinones have a weak redox cycle. This paper presents the results of the MTT test (primary screening for antitumor activity) of some 3-arylamino-1,4-naphthoquinone-4-oximes which were synthesized earlier. It has been shown that (4E)-4-(hydroxyimino)-3-(p-ethoxyphenylamino)naphtho-1(4H)-one and (4E)-4-(hydroxyimino)-3-(p-tolylamino)naphtho-1(4H)-one are most active against the colon adenocarcinoma and chronic myeloid leukemia in the tumor cell lines, respectively. We have performed the oximation reaction of 2-alkyl(benzyl)amino-1,4-naphthoquinones with hydroxylamine in ethanol to obtain a series of (4E)-3-alkyl(benzyl)amino-4-(hydroxyimino)naphthalene-1(4H)-ones. The reactions of synthesized products with 2,2-dihydroxy-1,3-indanedione (ninhydrin) have been studied under various conditions, i.e., in acetic acid under moderate heating and in dimethyl sulfoxide in the presence of methanesulfonic acid at different temperatures. It has been demonstrated that the interaction of (4E)-3-alkyl(benzyl)amino-4-(hydroxyimino)naphthalene-1(4H)-ones with ninhydrin in acetic acid leads to (6E)-5-alkyl(benzyl)amino-6-гhudroxyimino-4b,11b-dihydroxy-4b,5-dihydrobenzo[f]indeno[1,2-b]indol-11,12(6H,11bH)-diones. More severe conditions (dimethyl sulfoxide, methanesulfonic acid, and heating) leads to a mixture of products, the main of which are 1–alkyl-3'H-spiro[benzo[f]indole-2,1’-isobenzofuran]-3,3’,4,9(1H)-tetraones and 13-alkyl(benzyl)benzo[f]isochromeno[4,3-b]indole-5,7,12(13H)-triones. The yields of the products depend on the chosen temperature regime of the reaction. The structure of all synthesized compounds is confirmed by the physico-chemical methods, i.e., 1H and 13C NMR spectroscopy, IR spectroscopy, high-resolution mass spectrometry, spectrophotometry and elemental analysis. For citation: Gornostaev L.M., Rudenko D.S., Kirik S.D., Krasnov V.I. Interaction of (4E)-3-alkyl(benzyl)amino-4-(hydroxyimino)naphthalene-1(4H)-ones with 2,2-dihydroxy-1,3-indanedione. ChemChemTech [Izv. Vyssh. Uchebn. Zaved. Khim. Khim. Tekhnol.]. 2024. V. 67. N 5. P. 24-35. DOI: 10.6060/ivkkt.20246705.7015.

Bacterial stigmasterol degradation involving radical flavin delta-24 desaturase and molybdenum-dependent C26 hydroxylase

Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an oxygenase-independent sterol degradation pathway was discovered in a cholesterol-grown denitrifying bacterium Sterolibacterium (S.) denitrificans. It achieves hydroxylation of the unactivated primary C26 of the isoprenoid side chain to an allylic alcohol via a phosphorylated intermediate in a four-step ATP-dependent enzyme cascade. However, this pathway is incompatible with the degradation of widely distributed steroids containing a double bond at C22 in the isoprenoid side chain such as the plant sterol stigmasterol. Here, we have enriched a prototypical delta-24 desaturase from S. denitrificans, which catalyses the electron acceptor-dependent oxidation of the intermediate stigmast-1,4-diene-3-one (SDO) to a conjugated (22, 24)-diene. We suggest an α4β4 architecture of the 440 kDa enzyme, with each subunit covalently binding an FMN cofactor to a histidyl residue. As isolated, both flavins are present as red semiquinone radicals, which can be reduced by SDO but cannot be oxidized even with strong oxidising agents. We propose a mechanism involving an allylic radical intermediate in which two flavin semiquinones each abstract one hydrogen atom from the substrate. The conjugated delta-22,24 moiety formed allows for the subsequent hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase 2 (S26DH2). In conclusion, the pathway elucidated for delta-22 steroids achieves oxygen-independent hydroxylation of the isoprenoid side chain by bypassing the ATP-dependent formation of a phosphorylated intermediate.

Unraveling Eumelanin Radical Formation by Nanodiamond Optical Relaxometry in a Living Cell.

Defect centers in a nanodiamond (ND) allow the detection of tiny magnetic fields in their direct surroundings, rendering them as an emerging tool for nanoscale sensing applications. Eumelanin, an abundant pigment, plays an important role in biology and material science. Here, for the first time, we evaluate the comproportionation reaction in eumelanin by detecting and quantifying semiquinone radicals through the nitrogen-vacancy color center. A thin layer of eumelanin is polymerized on the surface of nanodiamonds (NDs), and depending on the environmental conditions, such as the local pH value, near-infrared, and ultraviolet light irradiation, the radicals form and react in situ. By combining experiments and theoretical simulations, we quantify the local number and kinetics of free radicals in the eumelanin layer. Next, the ND sensor enters the cells via endosomal vesicles. We quantify the number of radicals formed within the eumelanin layer in these acidic compartments by applying optical relaxometry measurements. In the future, we believe that the ND quantum sensor could provide valuable insights into the chemistry of eumelanin, which could contribute to the understanding and treatment of eumelanin- and melanin-related diseases.

A stable colorimetric biosensor for highly selective detection of malathion residue in food based on aptamer-regulated laccase-mimic activity

Malathion causes a serious threat to human health due to its widespread use in the environment. Herein, a novel and stable smartphone-integrated colorimetric biosensor for malathion detection is firstly established based on aptamer-enhanced laccase-mimicking activity. The results indicate that the M17-F aptamer can increase the affinity of Ag2O nanoparticles to the substrate 2,4-dichlorophenol and enhance their laccase-mimicking activity. Thus, abundant semiquinone radicals are produced in the catalytic system, which are combined with chromogenic agent to generate dark red products. The corresponding RGB values for the colour change of the solution can be easily obtained using smartphones, which is used for the rapid detection of malathion. The established biosensor for malathion has a limit of detection as low as 5.85 nmol·L−1, and displays good selectivity for other competitive pesticides. Moreover, further studies have verified the applicability of the biosensor in actual samples, indicating that it may have the potential for application in malathion detection in food.

Covalent conjugation of Inca peanut albumin and polyphenols with different phenolic hydroxyl numbers through laccase catalysis to improve functional properties.

Enzymatic crosslinking is a method that can be used to modify Inca peanut albumin (IPA) using polyphenols, and provides desirable functionalities; however, the effect of polyphenol structures on conjugate properties is unclear. In this study, we selected four polyphenols with different numbers of phenolic hydroxyl groups [para-hydroxybenzoic acid (HBA), protocatechuic acid (PCA), gallic acid (GA), and epigallocatechin gallate (EGCG)] for covalent modification of IPA by enzymatic crosslinking, and explored the structure-function changes of the IPA-polyphenol conjugates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis showed that laccase successfully promoted covalent crosslinking of IPA with polyphenols, with the order of degree of conjugation as EGCG > GA > PCA > HBA, the IPA-EGCG conjugate showed the highest polyphenol binding equivalents (98.35 g kg-1 protein), and a significant reduction in the content of free amino, sulfhydryl, and tyrosine group. The oxidation of polyphenols by laccase forms quinone or semiquinone radicals that are covalently crosslinked to the reactive groups of IPA, leading to significant changes in the secondary and tertiary structures of IPA, with spherical structures transforming into dense lamellar structures. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability and emulsification stability of IPA-EGCG conjugates improved by almost 6-fold and 2.7-fold, respectively, compared with those of unmodified IPA. These data suggest that the higher the number of polyphenol hydroxyl groups, the higher the degree of IPA-polyphenol conjugation; additionally, enzymatic crosslinking can significantly improve the functional properties of IPA. © 2024 Society of Chemical Industry.