Non-enzymatic Approaches Research Articles

An Overview of Interactions between Goat Milk Casein and Other Food Components: Polysaccharides, Polyphenols, and Metal Ions.

Casein is among the most abundant proteins in milk and has high nutritional value. Casein's interactions with polysaccharides, polyphenols, and metal ions are important for regulating the functional properties and textural quality of dairy foods. To improve the functional properties of casein-based foods, a deep understanding of the interaction mechanisms and the influencing factors between casein and other food components is required. This review started by elucidating the interaction mechanism of casein with polysaccharides, polyphenols, and metal ions. Thermodynamic incompatibility and attraction are the fundamental factors in determining the interaction types between casein and polysaccharides, which leads to different phase behaviors and microstructural types in casein-based foods. Additionally, the interaction of casein with polyphenols primarily occurs through non-covalent (hydrogen bonding, hydrophobic interactions, van der Waals forces, and ionic bonding) or covalent interaction (primarily based on the oxidation of proteins or polyphenols by enzymatic or non-enzymatic (alkaline or free radical grafting) approaches). Moreover, the selectivity of casein to specific metal ions is also introduced. Factors affecting the binding of casein to the above three components, such as temperature, pH, the mixing ratio, and the fine structure of these components, are also summarized to provide a good foundation for casein-based food applications.

Nonenzymatic electrochemical approaches for promethazine monitoring in aquatic media

Promethazine hydrochloride (PMH) is a phenothiazine derivative extensively utilized in healthcare settings. Its excessive consumption and continuous release into environmental water bodies can lead to adverse effects on human health and disrupt the equilibrium of aquatic ecosystems. Therefore, monitoring PMH levels is essential to maintain therapeutic efficacy and safeguard the well-being of both humans and aquatic life. This study encompasses the synthesis of samarium tungstate nanorods (SmWO) via hydrothermal method, followed by their incorporation into a composite with sulfur-doped graphitic carbon nitride (SmWO/SGCN) using ultrasonication technique. The morphology and physicochemical properties of the synthesized composite were thoroughly examined using various analytical methods. Subsequently, the electrocatalytic activity of the developed sensor was assessed for sensing PMH via voltammetry techniques. Under optimized conditions, the fabricated electrode exhibited an 8.1 nM detection limit with a wide linear range (0.02–199 µM), and high sensitivity (2.349 µA µM−1 cm−2) for PMH detection. The suggested SmWO/SGCN composite electrode shows promising practical potential for detecting PMH in environmental aquatic samples.

Metal-based non-enzymatic systems for cholesterol detection: mechanisms, features, and performance.

Metal based catalysts and electrodes are versatile tools known for their redox properties, catalytic efficiency, and stability under various conditions. Despite the absence of significant scientific hurdles, the utilization of these methods in cholesterol detection, particularly in non-enzymatic approaches, has been relatively underexplored. To this end, there is a pressing need to delve deeper into existing metal-based systems used in non-enzymatic cholesterol sensing, with the goal of fostering the development of innovative practical solutions. Various electrode systems, such as those employing Ni, Ti, Cu, Zn, W, Mn, and Fe, have already been reported for non-enzymatic cholesterol detection, some of them elucidated sensing mechanisms and potential in physiological detection. A detailed mechanistic understanding of oxide-based cholesterol sensors, along with the methodologies for constructing such systems, holds promise of advancing the exploration of practical applications. This review aims to provide a broad perspective on metal oxide systems and their characteristics that are conducive to non-enzymatic cholesterol sensing. It is intended to serve as a springboard with offering a guide to the design and development of efficient and sensitive electrochemical cholesterol sensors.

High-Precision Nonenzymatic Electrochemical Glucose Sensing Based on CNTs/CuO Nanocomposite

The measurement of blood glucose levels is essential for diagnosing and managing diabetes. Enzymatic and nonenzymatic approaches using electrochemical biosensors are used to measure serum or plasma glucose accurately. Current research aims to develop and improve noninvasive methods of detecting glucose in sweat that are accurate, sensitive, and stable. The carbon nanotube (CNT)-copper oxide (CuO) nanocomposite (NC) improved direct electron transport to the electrode surface in this study. The complex precipitation method was used to make this NC. X-ray diffraction (XRD) and scanning electron microscopy were used to investigate the crystal structure and morphology of the prepared catalyst. Using cyclic voltammetry and amperometry, the electrocatalytic activity of the as-prepared catalyst was evaluated. The electrocatalytic activity in artificial sweat solution was examined at various scan rates and at various glucose concentrations. The detection limit of the CNT-CuO NC catalyst was 3.90 µM, with a sensitivity of 15.3 mA cm−2 µM−1 in a linear range of 5–100 µM. Furthermore, this NC demonstrated a high degree of selectivity for various bio-compounds found in sweat, with no interfering cross-reactions from these species. The CNT-CuO NC, as produced, has good sensitivity, rapid reaction time (2 s), and stability, indicating its potential for glucose sensing.Graphical

Catalytic Kinetic Resolution and Desymmetrization of Amines

AbstractOptically active amines represent critically important subunits in bioactive natural products and pharmaceuticals, as well as key scaffolds in chiral catalysts and ligands. Kinetic resolution of racemic amines and enantioselective desymmetrization of prochiral amines have proved to be efficient methods to access enantioenriched amines, especially when the racemic or prochiral amines were easy to prepare while the chiral ones are difficult to be accessed directly. In this Account, we systematically summarized the development of kinetic resolution and desymmetrization of amines through nonenzymatic asymmetric catalytic approaches in the last two decades.1 Introduction2 Kinetic Resolution of Amines2.1 Kinetic Resolution of Amines via Asymmetric Transformations of the Amino Group2.1.1 Asymmetric N-Acylations2.1.2 Asymmetric N-Alkylation2.1.3 Asymmetric N-Arylation2.1.4 Other Asymmetric N-Functionalizations2.1.5 Asymmetric Dehydrogenation of Amines2.1.6 Selective C–N Bond Cleavage of Amines2.2 Kinetic Resolution of Amines via Asymmetric Transformations without Amino Group Participating3 Enantioselective Desymmetrization of Amines3.1 Desymmetrization of Diamines3.2 Desymmetrization of Prochiral Monoamines4 Conclusion and Outlooks

Recent Advances in Enzymatic and Non-Enzymatic Electrochemical Glucose Sensing.

The detection of glucose is crucial in the management of diabetes and other medical conditions but also crucial in a wide range of industries such as food and beverages. The development of glucose sensors in the past century has allowed diabetic patients to effectively manage their disease and has saved lives. First-generation glucose sensors have considerable limitations in sensitivity and selectivity which has spurred the development of more advanced approaches for both the medical and industrial sectors. The wide range of application areas has resulted in a range of materials and fabrication techniques to produce novel glucose sensors that have higher sensitivity and selectivity, lower cost, and are simpler to use. A major focus has been on the development of enzymatic electrochemical sensors, typically using glucose oxidase. However, non-enzymatic approaches using direct electrochemistry of glucose on noble metals are now a viable approach in glucose biosensor design. This review discusses the mechanisms of electrochemical glucose sensing with a focus on the different generations of enzymatic-based sensors, their recent advances, and provides an overview of the next generation of non-enzymatic sensors. Advancements in manufacturing techniques and materials are key in propelling the field of glucose sensing, however, significant limitations remain which are highlighted in this review and requires addressing to obtain a more stable, sensitive, selective, cost efficient, and real-time glucose sensor.

Decorated graphene oxide flakes with integrated complex of 8-hydroxyquinoline/NiO toward accurate detection of glucose at physiological conditions

Abstract The dramatic increase in the number of diabetic people raised the requirement for developing practical diagnostic setups capable of rapid and precise detection of glucose molecules at physiological conditions through non-enzymatic approaches. To address this requirement, we have developed a highly stable and sensitive non-enzymatic glucose biosensor consisted of decorated graphene oxide (GO) with an integrated platform of nickel oxide, 8-hydroxyquinoline (8H) and N-hydroxysuccinimide (NHS) (GO-NiO-8H-NHS) that enables the detection of glucose at neutral pH values. The optimum platform showed a perfect detection limit (DL) and sensitivity of 41 nM and 712.5 µA.mM−1.cm−2, respectively, via amperometric technique, while these values for voltammetric approaches were measured to be 63 nM and 4911.4 µA.mM−1.cm−2, respectively, at physiological conditions. Correspondingly, the developed platform showed superior sensitivity and accuracy toward detecting glucose within actual blood plasma samples and kept 94% of its initial performance after 30 days. Obtained outcomes revealed the fantastic DL and sensitivity of the developed non-enzymatic biosensors toward detecting glucose in actual biological samples at physiological conditions without using hazardous alkaline solutions.

Macrophage retrieval from 3D biomaterials: A detailed comparison of common dissociation methods

IntroductionThe immune system is widely recognized as a crucial determinant in implant biocompatibility and tissue integration. In order to assess macrophage response to biomaterials, commonly used analysis techniques require the removal of cells from the material. This process inherently has a negative impact on the cells, but few studies have comprehensively compared different dissociation methods in terms of impact on cell yield, viability, phenotype and function. MethodsCell scraping, EDTA at 4 °C, EDTA at 37 °C, trypsin, Accutase and the PromoCell macrophage detachment solution were compared in terms of cell yield and viability. The effect of Accutase on cell surface antigen conservancy and cell functionality was investigated. Lastly, effect of Accutase detachment of macrophages from electrospun biomaterials was analyzed. ResultsThe efficiency of common cell detachment protocols for human monocyte-derived macrophages (MDMs) varies significantly between enzymatic and non-enzymatic approaches. In terms of surface marker detection, we showed that enzymatic detachment not only selectively cleaves the M2 markers CD206 and CD163, but we also identified that this effect is variable across donors. Furthermore, we observed that the process of cell detachment impairs macrophage endocytic ability. Lastly, we tested the efficiency of enzymatic cell detachment on electrospun 3D matrices designed for tissue engineering. ConclusionOur results provide a thorough understanding of the advantages and disadvantages of commonly used cell dissociation methods and have important implications for studies investigating the macrophage response to biomaterials.

Microgel-Catalyzed Hydrolysis of Nonactivated Disaccharides

The controlled and selective hydrolysis of underivatized disaccharides and oligosaccharides remains a challenge that is met by enzymatic and nonenzymatic approaches. In an effort to capitalize on recent progress in the development of functional enzyme mimics for the hydrolysis of glycosidic bonds, we developed cross-linked microgels with embedded binuclear copper(II) complexes that are shown here to hydrolyze 1→4 over 1→6 glycosidic bonds under mildly alkaline conditions at elevated temperatures. The microgel catalysts show an unusual preference for the hydrolysis of 1→4β- over 1→4α-glycosidic bonds yielding up to 25 μg L–1 of glucose from cellobiose over 72 h and about half of that during the hydrolysis of maltose after correction for background effects. The experimental results are supported by computational analyses of the interactions between the embedded catalyst and the nonactivated disaccharide in putative transition state structures of the assembly during hydrolysis of the nonactivated glycosidic bond to rationalize this observation.

Structural analysis of holothurian fucosylated chondroitin sulfates: Degradation versus non-destructive approach

Fucosylated chondroitin sulfates (FCS) are unique glycosaminoglycans isolated from the body walls of sea cucumbers (holothuria). These biopolymers are composed of a chondroitin core [→4)-β-d-GlcA-(1 → 3)-β-d-GalNAc-(1→]n bearing fucosyl branches and the sulfate groups. Fucosyl substituents prevent enzymatic degradations of FCS by chondroitinases, which are effectively used in the structural analysis of vertebrate chondroitin sulfates, and hence, several non-enzymatic approaches to structural elucidation of FCS have been developed. They include combination of chemical modifications, specific degradation of polymeric chain and spectroscopic investigations of the products. In addition, there are examples of successful determination of the structure of non-modified FCS using NMR spectroscopic methods. NMR spectroscopy is especially effective in characterization of molecules with regular distribution of substituents along the polymeric chains. Otherwise combination of both chemical and spectroscopic methods is required for unambiguous elucidation of non-regular FCS structures. Structural variations of FCS studied up to now were shown to be species specific and include differences in type, amount and position of branches, as well as in degree and pattern of sulfation. These features of FCS can modify significantly the spectrum of their biological activities. In this mini-review the scopes and limitations of different approaches to FCS structure determination are concisely described and compared.

Direct Electrochemical Detection of Glutamate, Acetylcholine, Choline, and Adenosine Using Non-Enzymatic Electrodes.

Non-electroactive neurotransmitters such as glutamate, acetylcholine, choline, and adenosine play a critical role in proper activity of living organisms, particularly in the nervous system. While enzyme-based sensing of this type of neurotransmitter has been a research interest for years, non-enzymatic approaches are gaining more attention because of their stability and low cost. Accordingly, this focused review aims to give a summary of the state of the art of non-enzymatic electrochemical sensors used for detection of neurotransmitter that lack an electrochemically active component. In place of using enzymes, transition metal materials such as those based on nickel show an acceptable level of catalytic activity for neurotransmitter sensing. They benefit from fast electron transport properties and high surface energy and their catalytic activity can be much improved if their surface is modified with nanomaterials such as carbon nanotubes and platinum nanoparticles. However, a general comparison reveals that the performance of non-enzymatic biosensors is still lower than those that use enzyme-based methods. Nevertheless, their excellent stability demonstrates that non-enzymatic neurotransmitter sensors warrant additional research in order to advance them toward becoming an acceptable replacement for the more expensive enzyme-based sensors.

The first chemoenzymatic total synthesis of the phytotoxic nonenolide putaminoxin and its (5S,6E,9S)-diastereomer

The limitations of an alcohol dehydrogenase regarding the oxidative kinetic resolution of homoallylic alcohol containing alkyl chains were investigated, leading to a valuable building block for the total synthesis of phytotoxic nonenolide putaminoxin. The enzymatic approach towards the enantioenriched homoallylic alcohol was compared to classical, nonenzymatic approaches using asymmetric reagent controlled allyl additions and the obtained building block was used for the total synthesis of putaminoxin and its (5S,6E,9S)-diastereomer. After the spectroscopic analysis of the synthesized compounds, discrepancies were observed to already published data of isolated and synthesized putaminoxin. Therefore, a systematic comparison of NMR data was carried out. The result underlines the necessity of total synthesis for the absolute assignment of configuration.

Chemoenzymatic Total Synthesis of the Proposed Structures of Putaminoxins B and D.

Different enzymatic and nonenzymatic approaches were tested and compared to afford enantiopure homoallylic and allylic alcohols as building blocks in a total synthesis showcase. Thereby, highly enantioselective alcohol dehydrogenases and the P450 BM3 monooxygenase variant A74G L188Q were compared to classical asymmetric reagent-controlled allyl additions. Thus, the first total syntheses of the proposed structures for putaminoxins B/D and their respective enantiomers were accomplished. Detailed spectroscopic analysis of the newly synthesized compounds unraveled a discrepancy with respect to the reported structures of putaminoxins B/D. Furthermore, it was demonstrated that total synthesis is generally required for unequivocal assignment of configuration, because purely comparative NMR studies and judgment by analogy can lead to false predictions.

Flow cytometry analysis of inflammatory cells isolated from the sciatic nerve and DRG after chronic constriction injury in mice

BackgroundCellular responses to nerve injury play a central role in the pathogenesis of neuropathic pain. However, the analysis of site specific cellular responses to nerve injury and neuropathic pain is limited to immunohistochemistry staining with numerous limitations. New methodsWe proposed to apply flow cytometry to overcome some of the limitations and developed two protocols for isolation of cells from small specimens of the sciatic nerve and dorsal root ganglion (DRG) in mice. Results and comparasion with existingmethods We found that both the non-enzymatic and enzymatic approaches were highly effective in harvesting a sufficient number of cells for flow cytometry analysis in normal and pathological conditions. The total number of cells in the injury site of the sciatic and its DRGs increased significantly 14days after chronic constriction injury (CCI) of the sciatic nerve, compared to sham surgery control or the contralateral control. The enzymatic approach yielded a significantly higher total number of cells and CD45 negative cells, suggesting that this approach allows for harvest of more resident cells, compared to the non-enzymatic method. The percentage of CD45+/CD11b+ cells was significantly increased in the sciatic nerve but not in the DRG. These results were consistent with both protocols. ConclusionsWe thus offer two simple and effective protocols that allow for application of flow cytometry to the investigation of cellular and molecular mechanisms of neuropathic pain.

Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy.

BackgroundRapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence microscopy and nucleic acid analogues have been proposed so far.Methods and ResultsHere we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least 1000-fold above the potential detection limit.ConclusionOverall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay, which allows for an accurate estimation of mutant abundance when the detection limit requirement is met. Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly in the biological sample of choice.

ChemInform Abstract: Chemistry and Biology of the Ubiquitin Signal

AbstractReview: 216 refs.

Nonenzymatic assembly of natural polyubiquitin chains of any linkage composition and isotopic labeling scheme.

Polymeric chains made of a small protein ubiquitin act as molecular signals regulating a variety of cellular processes controlling essentially all aspects of eukaryotic biology. Uncovering the mechanisms that allow differently linked polyubiquitin chains to serve as distinct molecular signals requires the ability to make these chains with the native connectivity, defined length, linkage composition, and in sufficient quantities. This, however, has been a major impediment in the ubiquitin field. Here, we present a robust, efficient, and widely accessible method for controlled iterative nonenzymatic assembly of polyubiquitin chains using recombinant ubiquitin monomers as the primary building blocks. This method uses silver-mediated condensation reaction between the C-terminal thioester of one ubiquitin and the ε-amine of a specific lysine on the other ubiquitin. We augment the nonenzymatic approaches developed recently by using removable orthogonal amine-protecting groups, Alloc and Boc. The use of bacterially expressed ubiquitins allows cost-effective isotopic enrichment of any individual monomer in the chain. We demonstrate that our method yields completely natural polyubiquitin chains (free of mutations and linked through native isopeptide bonds) of essentially any desired length, linkage composition, and isotopic labeling scheme, and in milligram quantities. Specifically, we successfully made Lys11-linked di-, tri-, and tetra-ubiquitins, Lys33-linked diubiquitin, and a mixed-linkage Lys33,Lys11-linked triubiquitin. We also demonstrate the ability to obtain, by high-resolution NMR, residue-specific information on ubiquitin units at any desired position in such chains. This method opens up essentially endless possibilities for rigorous structural and functional studies of polyubiquitin signals.