Peroxidase Substrate Research Articles

Measuring Myeloperoxidase Activity as a Marker of Inflammation in Gut Tissue Samples of Mice and Rat.

Myeloperoxidase (MPO) is an enzyme contained in lysosomal azurophilic granules of neutrophils. MPO activity has been shown to correlate with the number of neutrophils in histological sections of the gastrointestinal tract and is therefore accepted as a biomarker of neutrophil invasion in the gut. This protocol describes an easy, cost-effective kinetic colorimetric assay to quantify myeloperoxidase activity in intestinal tissue samples. It is explained using tissue collected in mice but can also be used for other laboratory animals. In a first step, tissue specimens are homogenized using a phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (HTAB), which extracts MPO from neutrophils. The obtained supernatant is added to a reagent solution containing o-dianisidine dihydrochloride, which is a peroxidase substrate. Finally, the change in absorption is measured via spectrophotometry and converted to a standardized unit of enzyme activity. The assay is illustrated and compared to a commercially available enzyme-linked immunoassay (ELISA), demonstrating that MPO activity does not necessarily correlate with MPO protein expression in tissue samples. Key features Optimized for use in mice and rats but can also be used for samples of other species. Measures enzymatic activity instead of mRNA or protein expression. Requires a spectrophotometer. Can be performed in duplo using 10 mg of (dry-blotted) gut tissue or more. Graphical overview.

Estimation of L-carnitine levels in diabetic completely edentulous patients for implant diagnosis: A cross-sectional study

Background: Carnitine is effective in preventing the accumulation of end products related to lipid peroxidation due to its anti-inflammatory and antioxidant effects. Carnitine also exerts a significant anti-inflammatory role through the downregulation of the nuclear factor kappa beta pathway, which leads to a decrease in the expression of pro-inflammatory cytokines. The aim of the study was to estimate the L-carnitine (L-C) levels in diabetic completely edentulous patients. Materials and Methods: A cross-sectional study was conducted after the selection of 60 samples based on the inclusion and exclusion criteria. The collected saliva samples were utilized to measure the levels of L-C using the sandwich enzyme-linked immunosorbent assay (ELISA) method. One hundred microliters of sample was applied to a particular row of wells and incubated for an hour as part of the sandwich ELISA procedure. After the wells had been cleaned, a second batch of monoclonal L-C was added, and they were once more incubated for an hour. The horseradish peroxidase substrate was then applied after washing the second batch as well. To allow the blue-to-yellow color transition, the wells were kept steady. Following the observation of the color shift, the OD was measured, and the concentration was determined using the sandwich ELISA kit's standard curve as an intercept. The data were statistically analyzed using the independent t-test (significant level P < 0.05) and were tabulated. Results: The L-C levels have higher levels in nondiabetic patients than in diabetic patients. The difference in the baseline mean value between the groups was statistically significant (P = 0.00). Although it is statistically significant (P = 0.00), the mean value for diabetic individuals is 0.19 as opposed to 0.29 for nondiabetic patients. Conclusion: Based on the findings, it can be concluded that L-C improves insulin sensitivity and glucose disposal in diabetic completely edentulous patients.

Lipid Peroxidation and Iron Metabolism: Two Corner Stones in the Homeostasis Control of Ferroptosis

Regulated cell death (RCD) has a significant impact on development, tissue homeostasis, and the occurrence of various diseases. Among different forms of RCD, ferroptosis is considered as a type of reactive oxygen species (ROS)-dependent regulated necrosis. ROS can react with polyunsaturated fatty acids (PUFAs) of the lipid (L) membrane via the formation of a lipid radical L• and induce lipid peroxidation to form L-ROS. Ferroptosis is triggered by an imbalance between lipid hydroperoxide (LOOH) detoxification and iron-dependent L-ROS accumulation. Intracellular iron accumulation and lipid peroxidation are two central biochemical events leading to ferroptosis. Organelles, including mitochondria and lysosomes are involved in the regulation of iron metabolism and redox imbalance in ferroptosis. In this review, we will provide an overview of lipid peroxidation, as well as key components involved in the ferroptotic cascade. The main mechanism that reduces ROS is the redox ability of glutathione (GSH). GSH, a tripeptide that includes glutamic acid, cysteine, and glycine, acts as an antioxidant and is the substrate of glutathione peroxidase 4 (GPX4), which is then converted into oxidized glutathione (GSSG). Increasing the expression of GSH can inhibit ferroptosis. We highlight the role of the xc- GSH-GPX4 pathway as the main pathway to regulate ferroptosis. The system xc-, composed of subunit solute carrier family members (SLC7A11 and SLC3A2), mediates the exchange of cystine and glutamate across the plasma membrane to synthesize GSH. Accumulating evidence indicates that ferroptosis requires the autophagy machinery for its execution. Ferritinophagy is used to describe the removal of the major iron storage protein ferritin by the autophagy machinery. Nuclear receptor coactivator 4 (NCOA4) is a cytosolic autophagy receptor used to bind ferritin for subsequent degradation by ferritinophagy. During ferritinophagy, stored iron released becomes available for biosynthetic pathways. The dysfunctional ferroptotic response is implicated in a variety of pathological conditions. Ferroptosis inducers or inhibitors targeting redox- or iron metabolism-related proteins and signal transduction have been developed. The simultaneous detection of intracellular and extracellular markers may help diagnose and treat diseases related to ferroptotic damage.

Platinum-Group Metal Nanoparticles as Peroxidase Mimics: Implications for Biosensing

Over the past few decades, peroxidase mimics made of platinum-group metal nanoparticles (PGM NPs) have been actively developed and applied to various biosensing platforms. Nevertheless, there is a lack of a comprehensive study that compares the peroxidase-like activities of PGM NPs and their performance in biosensing. Here, we report a systematic study of PGM NPs as peroxidase mimics, including Pd, Pt, Rh, and Ir NPs. NPs of these elements were uniformly synthesized and their nanoscale features were probed to ensure a consistent size, shape, and chemical ligand on the surface. Our measurements indicate that the Ir NP is the most active one with a catalytic constant as high as 6.27 × 105 s–1, followed by Pt, Rh, and Pd NPs. The binding affinities of NPs to peroxidase substrates during catalysis were also quantitively analyzed and compared. Using enzyme-linked immunosorbent assay as a model biosensing platform, the performance of PGM NPs in detecting carcinoembryonic antigen (a cancer biomarker) was evaluated. The results showed that the detection sensitivity was correlated to the catalytic activity of PGM NPs, where Ir NPs achieved the highest sensitivity with a limit of detection at the level of low picogram per milliliter.

Facile fabrication of metal‐organic frameworks with peroxidase‐like activity for the colorimetric detection of Alicyclobacillus acidoterrestris

AbstractAlicyclobacillus acidoterrestris (A. acidoterrestris), a kind of spore‐forming bacterium, could causes spoilage of fruit juices, which results in pecuniary losses to the industry. Unfortunately, the conventional methods used to identify this bacterium are laborious and complex. In this work, copper‐based metal‐organic frameworks (Cu‐MOFs) with intrinsic peroxidase‐like activity were fabricated by a self‐assembly method. Based on the properties of Cu‐MOFs, a colorimetric assay was developed for the detection of guaiacol, a characteristic metabolite of A. acidoterrestris and chromogenic substrate of peroxidase. Under optimal conditions, an excellent linear relationship was achieved over a range of guaiacol (0.01–0.4 mM), with a limit of detection (LOD) as low as 0.003 mM. This biosensor also showed a satisfactory sensing performance towards guaiacol, allowing the identification of A. acidoterrestris with high selectivity in a real sample. This present study provides a new perspective for the exploration of more sensitive and convenient methods for A. acidoterrestris detection.

Catalytic Gold-Iridium Nanoparticles as Labels for Sensitive Colorimetric Lateral Flow Assay.

The colorimetric lateral flow assay (CLFA, also known as test strip) is a widely used point-of-care diagnostic technology. It has been a challenge to significantly improve the detection sensitivity of CLFA without involving additional equipment and/or compromising its simplicity. In this work, we break through the detection limit barrier of CLFA by developing a type of catalytic nanoparticles (NPs) used as labels. Specifically, the NPs were engineered by coating conventional gold NPs (AuNPs) with iridium (Ir) to form an Au-Ir core-shell structure. Such Au-Ir NPs possess ultrahigh peroxidase-like catalytic activities. A single Au-Ir NP can generate up to 107 colored molecules per second by catalyzing peroxidase substrates. The strong color signal from the catalysis ensures a high sensitivity of associated CLFA. The Au-Ir NP-based CLFA was successfully applied to the detection of two different cancer biomarkers that achieved limits of detection at the low picogram per milliliter level, hundreds of times lower than those of conventional AuNP-based CLFA.

Evaluation of chromogenic substrates for horseradish peroxidase on paper-based microfluidic devices

Microfluidic paper-based analytical devices (microPADs) are an emerging platform for point-of-care assays with potential applications ranging from medical diagnostics to environmental monitoring. A standard approach for signal generation and amplification on microPADs is the use of enzymes and chromogenic substrates, which produce colored products and enable the qualitative and quantitative detection of analytes. Horseradish peroxidase (HRP) is one of the most commonly used enzymes for this application. While a wide variety of chromogenic substrates and substrate solutions are available for HRP, these were all originally developed and optimized for other platforms and have not been systematically compared on microPADs. In this work, 33 chromogenic substrate solutions for HRP containing one of six different substrate molecules were evaluated in a standardized colorimetric assay and compared on the basis of limit of detection (LOD), lower limit of quantitation (LLOQ), upper limit of quantitation (ULOQ), sensitivity, dynamic range, stability, and cost. While the substrates tetramethylbenzidine (TMB) and o-phenylenediamine (OPD) were found to achieve the best overall analytical performance, each substrate had characteristics that may make them appealing for specific applications. The results of this work will facilitate the selection of substrate molecules and the formulation of substrate solutions for future colorimetric paper-based assays involving HRP.

Novel Colorimetric Aptasensor for the Detection of Golgi Protein 73 by Exploiting the Peroxidase-Like Activity of the H-rGO-Mn3O4 Nanozyme

Golgi protein 73 (GP73) is a novel biomarker for the detection of hepatocellular carcinoma. We prepare a colorimetric aptasensor for GP73 detection based on the catalytic performance of the H-rGO-Mn3O4 nanozyme. The H-rGO-Mn3O4 nanozyme serves as a peroxidase mimetic which can enhance the reaction with the substrate (3, 3,’ 5, 5′-tetramethylbenzidine, TMB) and H2O2. The peroxidase-like activity of H-rGO-Mn3O4 features a 1.78-fold higher binding affinity value (Km) for TMB compared with that of horseradish peroxidase. The H-rGO-Mn3O4 nanozyme not only exhibits admirable peroxidase-like activities due to the synergistic effect of Mn3O4 NPs, rGO and hemin, but also has a large specific surface area to endow the GP73 aptamer with specific recognition capabilities. A sandwich colorimetric aptasensor is formed to realize the visual detection of GP73 through catalyzing the H2O2-mediated oxidation peroxidase substrate TMB to oxidized TMB accompanied by a color change from colorless to blue. Under optimal conditions, the logarithm of the GP73 concentration (0.05–50.0 ng/mL) shows a good linear relationship with the absorbance. The calibration equation is Y =−0.0383 lgC+0.4835, with R2 of 0.9964, and the lowest limit of detection is 36.94 pg/mL. Additionally, the content of GP73 in human serum samples is directly detected, and the relative standard deviation is 0.49–4.91%. Compared with the enzyme-linked immunosorbent assay method, the relative error is 0.23–3.61%. All in all, colorimetric aptasensor is demonstrated to exhibit excellent specificity, stability, and reproducibility.

A Facile Preparation of Reduced Graphene Oxide Capped AuAg Bimetallic Nanoparticles: A Selective Nanozyme for Glutathione Detection

AbstractOver the last decade, artificial enzymes (nanozymes) have undergone substantial development in the field of sensing. In this study, Au−Ag bimetallic nanoparticles (NPs) decorated on reduced graphene oxide sheets (AuAg‐rGO) nanocomposite has been developed as a novel colorimetric probe based on the peroxidase imitating activity of the nanozyme. AuAg‐rGO nanocomposite proved to feature an intrinsic peroxidase mimic activity, facilitating the oxidation of a peroxidase substrate like 3,3’,5,5’‐tetramethylbenzidine (TMB) with high efficiency to oxidised TMB (blue coloured product) with the aid of hydrogen peroxide (H2O2). The characteristic Michaelis‐Menten kinetics study allowed the establishment of the catalytic performance of AuAg‐rGO as an artificial enzyme. Glutathione (GSH) displays inhibiting effect on the oxidation of the peroxidase substrate, TMB. Therefore, AuAg‐rGO nanozyme was applied for detection of GSH and exhibited exceptional selectivity and sensitivity with a low limit of detection (LOD) of 38 nM within the 0–200 μM concentration range. The artificial nanozyme system reported here is simple, convenient, and straightforward for colorimetric sensing of GSH.

Synthesis of silver decorated magnetic Fe3O4/alginate polymeric surfactant with controllable catalytic activity toward p-NP removal and enzymatic-mimic activity for solid-colorimetric H2O2 detection

In this study, a self-assembly method has been employed for providing a hybrid silver magnetite nanocomposite with superior catalytic and enzymatic peroxidase mimic activity. Interestingly, the tail variation of a newly synthesized polymeric surfactant showed a controlling impact in improving the catalytic activity of the silver-magnetic nanocomposite as a unique study. The hybrid composites were characterized via TEM, SEM, and XRD, revealing the successful preparation of self-assembled AgNPs on the magnetite Fe3O4 nanocomposite surface. The hydrophobic chain of the surfactant improved the catalytic performance of both bare magnetic Fe3O4, and also the silver decorated hybrid magnetic nanocomposite Fe3O4@AMALG8/12@Ag. The decorated Fe3O4@AMALG12@Ag with longest hydrophobic tail exhibited higher catalytic reduction activity to convert p-nitrophenol (p-NP) into p-aminophenol (p-AP) than Fe3O4@AMALG18@Ag which prepared using shorter hydrophobic tail. The engineered hybrid magnetic nanocomposite exhibited super peroxidase mimic activity compared to bare one toward converting the colorless o-phenylenediamine (OPD) into the yellow 2, 3-diaminophenazine (DAP). In the same context, the surfactant participated significantly in improving the peroxidase activity as well. Ultimately, the decorated Fe3O4@AMALG12@Ag was integrated in agarose hydrogel structure with OPD as a peroxidase substrate for colorimetric sensing the H2O2. The solid kit exhibited 0 to 1250 μM as a linear dynamic range with low limit detection (LOD) of 14 μM. The developed solid kit could be applied for detecting different biomarkers such as glucose, lactate, uric and cholesterol based on selective enzymatic release of H₂O₂.

Rational Design of Conducting Polymer-Derived Tubular Carbon Nanoreactors for Enhanced Enzyme-like Catalysis and Total Antioxidant Capacity Bioassay Application.

The design of void-confined tubular nanostructures has aroused significant interest for catalytic applications because of their distinct microenvironment to modulate the reaction kinetics. Herein, we propose a facile wrapping-pyrolysis strategy to confine Fe0 nanoparticles (Fe NPs) inside N-doped carbon nanotubes (Fe@NC NTs) derived from Fe2O3@polypyrrole (PPy) core-sheath nanofibers (NFs). The resultant Fe@NC NTs can act as efficient enzyme mimics and exhibit a significantly higher peroxidase (POD)-like catalytic activity than unconfined Fe NPs and bare NC NTs. Kinetic experiments demonstrate that the optimized void structure benefits the affinity with the POD substrates and achieves excellent catalytic efficiency. The mechanism study reveals that the generation of •OH from H2O2 endows Fe@NC NTs with excellent POD-like performance. Furthermore, we develop a total antioxidant capacity (TAC) sensing platform on account of this efficient POD-like system, expanding their applications in the field of food safety and human healthcare.

Peroxidase‐like activity of Fe3O4 nanoparticles and Fe3O4‐graphene oxide nanohybrids: Effect of the amino‐ and carboxyl‐surface modifications on H2O2 sensing

Magnetic, iron oxide nanoparticles and graphene‐iron oxide nanohybrids have been introduced as artificial peroxidase‐mimics. In the present paper, the impact of coating with different functional groups on the peroxidase activity of both nanoparticles and nanohybrids was studied. Magnetite nanoparticles and graphene‐iron oxide nanohybrids, with ‐amino or ‐carboxyl terminal groups were synthesized and fully characterized by Fourier transform‐infrared, thermogravimetric analysis, transmission electron microscopy (TEM), X‐ray diffraction, vibrating sample magnetometer, and zeta potential. All particle formulations were evaluated for peroxidase activity, by studying the oxidation reaction of the peroxidase substrate 3,3,5,5‐tetramethylbenzidine (TMB) by hydrogen peroxide (H2O2). It was found that all tested formulations retained their catalytic ability (>70%) at 95 °C, at wide pH range, and were reusable with 60% of their catalytic activity after 10 uses. All nanomaterials were found to follow Michaelis–Menten kinetics and peroxidase activity varied with respect to electrostatic affinity between nanoparticles or nanohybrids and the peroxidase substrates, evidenced by differences in determined kinetic parameters. The Km values of all tested particles with H2O2 substrate were higher than those reported for horseradish peroxidase (HRP; 0.214–6.36 mM), while the calculated values of reaction rates (Vmax) for the studied nanoparticles and nanohybrids with TMB and H2O2 as substrates were comparable with those reported for HRP. Our findings confirm that iron oxide nanoparticles and graphene based metallic nanohybrids can be tailor‐made to possess improved peroxidase‐like activity. Such enhancements could further widen nanohybrids application in a wide range of biomedical applications, including H2O2 detection in a proof‐of‐concept assay, which confirmed low detection limits of the tested formulations (5.3–11.3 μM).

Biocompatible pericarpium citri reticulatae polysaccharide templated Pd nanoparticles for effectively colorimetric detection of glutathione

Glutathione as a natural antioxidant can protect the human body from free radicals produced by oxidative stress. Therefore, it’s quite necessary to measure the concentration of glutathione to ensure our health. Herein, we prepared biocompatible pericarpium citri reticulatae polysaccharide templated palladium nanoparticles (Pdn-PCRP NPs, n = 150, 225, 300) by a green and facile synthetic method. The sizes of Pd150-PCRP NPs, Pd225-PCRP NPs, and Pd300-PCRP NPs were 2.10 ± 0.62 nm, 2.48 ± 0.62 nm, and 2.63 ± 0.62 nm, respectively. Pd150-PCRP NPs exhibited excellent biocompatibility based on the MTT assay. Moreover, Pd150-PCRP NPs oxidized the colorless peroxidase substrates 3,3′,5,5′ -tetramethylbenzidine (TMB) to its corresponding blue product in the presence of hydrogen peroxide. More importantly, Pd150-PCRP NPs were used in the colorimetric detection of glutathione. The detection range of colorimetric detection with Pd150-PCRP NPs reached 2–300 μM and the detection limit was 1.65 μM. The recovery rate of glutathione in serum samples was as high as 103.60 %. Overall, these results demonstrated that Pd150-PCRP NPs had good biocompatibility, peroxidase-like activity and ability of glutathione detection. Pd150-PCRP NPs could serve as a safe and promising agent for sensitive detection of glutathione in future applications

RuO2/rGO heterostructures as mimic peroxidases for colorimetric detection of glucose.

Thesuccessful synthesis of ruthenium oxide/reduced graphene oxide (RuO2/rGO) heterostructures by one-pot hydrothermal method using graphene oxides and RuCl3 as precursorsis reported. The heterostructures had high peroxidase-like (POD-like) activities, which catalyzes the oxidation of classical peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2 to create a blue colored reaction product. The catalytic activity was significantly enhanced by the synergistic effect between RuO2 nanoparticles and rGO. RuO2/rGO had a low Km of 0.068mM and a high vmax of 1.228 × 10-7 M·s-1 towards TMB in theTMB-H2O2 catalytic oxidation system. In addition, the POD-like activity originating from the electron transfer mechanism was confirmed by cytochrome C (Cyt C) oxidation experiment. A colorimetric method based on RuO2/rGO heterostructures was developed with good sensitivity and selectivity for glucose detection with a limit of detection of 3.34μM and a linear range of 0-1500μM. The RuO2/rGO heterostructures havepotential applications in the biomedical areas, such as biosensor and diagnostics.

Uric Acid Reacts with Peroxidasin, Decreases Collagen IV Crosslink, Impairs Human Endothelial Cell Migration and Adhesion.

Uric acid is considered the main substrate for peroxidases in plasma. The oxidation of uric acid by human peroxidases generates urate free radical and urate hydroperoxide, which might affect endothelial function and explain, at least in part, the harmful effects of uric acid on the vascular system. Peroxidasin (PXDN), the most recent heme-peroxidase described in humans, catalyzes the formation of hypobromous acid, which mediates collagen IV crosslinks in the extracellular matrix. This enzyme has gained increasing scientific interest since it is associated with cardiovascular disease, cancer, and renal fibrosis. The main objective here was to investigate whether uric acid would react with PXDN and compromise the function of the enzyme in human endothelial cells. Urate decreased Amplex Red oxidation and brominating activity in the extracellular matrix (ECM) from HEK293/PXDN overexpressing cells and in the secretome of HUVECs. Parallelly, urate was oxidized to 5-hydroxyisourate. It also decreased collagen IV crosslink in isolated ECM from PFHR9 cells. Urate, the PXDN inhibitor phloroglucinol, and the PXDN knockdown impaired migration and adhesion of HUVECs. These results demonstrated that uric acid can affect extracellular matrix formation by competing for PXDN. The oxidation of uric acid by PXDN is likely a relevant mechanism in the endothelial dysfunction related to this metabolite.

Immunofluorescent and Immunohistochemical (Colorimetric) Characterization of B‐DNA, Z‐DNA and G4‐Quadruplex DNA in Human Tissues Demonstrating the Spatial Genomic Organization of Different DNA Structures: Genomesorganizomics

Spatial genomic organization, relative to DNA and RNA structures, has not been examined. DNA can adopt a variety of different shapes (helical conformations), i.e., canonical right‐handed double‐stranded (ds‐) B‐DNA, alternative left‐handed ds‐Z‐DNA and multi‐stranded G4‐quadruplex DNA. Each of these DNAs has a unique structure and plays a different role in cells. The structure of the nucleus is complex, and has a major role in gene expression. For the first time, our group has demonstrated the spatial genomic distribution of three totally different DNAs concurrently in HeLa cells, paraffin‐embedded tissue sections, and frozen sections. This was achieved by means of anti‐B‐DNA, anti‐Z‐DNA and anti‐G4 quadruplex DNA antibodies, via immunofluorescence (IF), and immunohistochemistry (IHC) (colorimetric). No one is investigating the relationship between spatial genomic organization and structures of different nucleic acids. All three types of DNA exist in the human genome and perform diverse cellular genomic functions. Tissues were processed in 10% NBF, and staining protocols were done both manually and by autostainer. Antigen retrieval enhanced all DNA‐based immunostaining procedures. IF was performed using Alexa Fluor conjugated secondary antibodies [i.e., Alexa Fluor 405 (blue), Alexa Fluor 488 (green) and Alexa Fluor 555 (red)], and wheat germ agglutinin conjugated with Alexa Fluor 647 (white) (counterstain). IHC protocols (i.e., enzyme: HRP or AP) were accomplished by using enzymes conjugated to secondary antibodies, and color developed by chromogens [i.e., B‐DNA (Blue) AP Substrate, Z‐DNA (Red/Magenta) AP Substrate, and G4‐quadruplex DNA (Brown), DAB Peroxidase (HRP) Substrate]. Negative and positive controls, isotype controls, and blocking background were performed in triplicate. The order of primary antibodies (and substrates: IHC) is critical for obtaining accurate data. Images were obtained by confocal and light microscopes (i.e., 200x, 600x). Advanced computer‐based image analysis (e.g., HALO, Image Pro Plus) allowed for qualitative and quantitative characterization of immunostained DNAs. The largest percentage of DNA was in the form of B‐DNA, followed by the other two remaining structures of DNA. The novel method signifies a new subfield of structural and functional genomics, which we have named Spatial Genomic Organization of DNA Structures, i.e., Genomesorganizomics. It will aid spatial transcriptomics in the analysis of gene expression and provide information on transcriptional activity based upon the conformation of DNA in genes and telomeres. An in‐depth comparative analysis between IF and IHC will yield information about specific advantages and disadvantages of these immuno‐based tissue staining systems (e.g., sensitivity, dynamic range, easier multiplexing, photobleaching, better target colocalization). This genomics approach is both sensitive and reliable, and will allow for the characterization of nuclear genomic architecture. Future projects are examining both DNA and RNA structures of four or more nucleic acids.

Faster and sensitive zymographic detection of oxidases generating hydrogen peroxide. The case of diamine oxidase

The existing zymography method for the detection of diamine oxidase (DAO) activity has been improved by a new staining procedure with the aim to ameliorate its sensitivity. Both procedures used SDS-PAGE gels containing uniformly distributed entrapped peroxidase (that wouldn't migrate during electrophoresis). The new approach with 3,5-dichloro-2-hydroxybenzenesulfonate (DCHBS) as peroxidase substrate and with 4-amino-antipyrine as color stabilizer allows a more sensitive detection of DAO when compared to the previously reported o-phenylenediamine (o-PDA) as peroxidase substrate. The newly improved method appears faster, simple and environmentally friendly. It can be used for most of oxidases releasing hydrogen peroxide as reaction product.

Ultrabright silicon nanoparticle fluorescence probe for sensitive detection of cholesterol in human serum.

A highly sensitive fluorescence-based assay for cholesterol detection was developed using water-dispersible green-emitting silicon nanoparticles (SiNPs) as a fluorescence indicator and enzyme-catalyzed oxidation product PPDox (Bandrowski's base) as a quencher. The SiNPs were facilely synthesized via a simple, one-step hydrothermal treatment using 3-[2-(2-aminoethylamino)ethylamino]propyl-trimethoxysilane (AEEA) as the silicon source, which has ultrahigh quantum yield and low phototoxicity. Under the catalysis of cholesterol oxidase (ChOx), hydrogen peroxide (H2O2) was generated as a result of cholesterol oxidation. Utilizing p-phenylenediamine (PPD) as the substrate for horseradish peroxidase (HRP) in the presence of H2O2 led to the production of PPDox. Based upon the inner filter effect (IFE), the established ultrasensitive fluorescent assay could accurately measure cholesterol. The limit of detection (LOD) of the assay was 0.018μM with a linear range of 0.025-10μM. The results for the detection of real serum samples by the proposed assay were comparable to those by a commercial reagent kit, demonstrating that our proposed strategy has high application potential in disease diagnosis and other related biological studies.

Indirect colorimetric determination of trace hydrogen peroxide by its oxidizing power on chromium(III) oxide nanoparticles

Although enzymes are excellent catalysts in vivo, they are difficult to use in vitro. Therefore, enzyme-mimetic nanoparticles (NPs) or nanozymes, having the properties of certain enzymes but being much more resistant to chemicals and atmospheric conditions, offer a good alternative to biological enzymes. In the present study, Cr2O3 NPs were used as redox catalysts for determination of H2O2, a key compound for biochemistry, food industry and environmental engineering. This method proved to be more selective for H2O2 than other similar nanosensing methods based on Au/Ag NPs formation. This simple method had high selectivity toward H2O2 due to the high redox potential of Cr(VI)/Cr(III) couple, which could not be overcome by mild oxidants. Unlike most studies in the literature using peroxidase-like nanozymes for colorimetric H2O2 determination, a peroxidase substrate is not required in this study. Here, when H2O2 in the real samples (or in case of triacetone triperoxide (TATP) samples, H2O2 released by hydrolysis) were contacted with Cr2O3 NPs at pH 10, Cr(III) was oxidized to Cr(VI). At optimal temperature and time, there was a clear reaction with a net stoichiometry between the probe and analyte. Finally, the generated Cr(VI) was reacted by diphenylcarbazide (DPC) in acidic medium to form a pink colored compound, the color intensity of which was directly related to H2O2 concentration. This method yielded a linear calibration range of 4.0–58 µM for H2O2. For the tested real samples and TATP hydrolyzate, the results obtained by the proposed procedure were compatible with those of the standard TiOSO4 colorimetric method. Finally, H2O2 scavenging effects of certain antioxidants (AOX) were successfully measured by the presented spectrophotometric Cr2O3 NPs method.

Peroxidase-like activity of hemoglobin-based hybrid materials against different substrates and their enhanced application for H2O2 detection

ABSTRACT. Organic-inorganic hybrid nanoflowers method with unique properties are preferred than conventional immobilization methods for the past decade. Hereemoglobin-based hybrid material (HbNFs@Cu) was synthesized under different experimental conditions (pH 5.0-9.0 and 0.01-0.50 mgmL-1 of hemoglobin) obtaining a material size of 9-10 µm. The encapsulation percentage and weight yield of HbNFs@Cu were determined as 100% and 6.7%, respectively. The peroxidase-like activities of the material against different substrates (ABTS and Guaiacol) were compared to free hemoglobin. The HbNFs@Cu hybrid structure exhibited Vmax of 3.6995 EU/mg and a Michaelis-Menten constant (KM) of 0.1357 mM/mL. The HbNFs@Cu hybrid material was then used to catalyze the oxidation of a peroxidase substrate ABTS to the pigmented product, which provided a colorimetric and spectrophotometric detection of H2O2. The linear operating range, detectable colorimetrically as H2O2 sensor, is 0.005-0.0042 mM, while the linear operating range, detectable spectrometically, is 0.003-0.0042 mM. The limits of detection of colorimetric and spectrophotometric sensors were 0.005 mM and 0.003 mM, respectively. Collectively, these results showed that HbNFs@Cu can be used as colorimetric biosensor for H2O2 in potential applications such as pharmaceutical food, biomedical, environmental, and industrial.
 
 KEY WORDS: Hydrogen peroxide, Hemoglobin, Hybrid Material, Colorimetric assay
 
 Bull. Chem. Soc. Ethiop. 2021, 35(3), 537-550. 
 DOI: https://dx.doi.org/10.4314/bcse.v35i3.6