A ratiomectic aptasensor with enhanced signals based on peroxidase-like enzymes and NH2-MIL-101@MoS2 for trace detection of deoxynivalenol in traditional Chinese herbs.

The accumulation of the deoxynivalenol (DON) in the human body poses a significant health risk that is often overlooked, and we urgently need an ultra-sensitive rapid detection platform. Due to the porosity of NH2-MIL-101@MoS2, an increased loading of toluidine blue (TB) serves to create a signal reference. Cobalt@carbon (CoC) derived from metal organic frameworks was combined with NH2-MIL-101(NH2-MIL-101@CoC) to form an enzyme-free Nanoprobe (Apt-pro) with significant catalytic properties. The ratio (IBQ /ITB) was changed by varying the electrochemical signal of benzoquinone (BQ) (IBQ) and the amount of TB deposition (ITB). This aptasensor was successfully applied to detect DON in malt and peach seed, which exhibited a great linear range from 1 fg/mL to 10 ng/mL and low detection limit of 0.31 fg/mL for DON.

Label-Free Detection of Epinephrine Using Flower-like Biomimetic CuS Antioxidant Nanozymes.

A biosensor comprising crystalline CuS nanoparticles (NPs) was synthesized via a one-step simple coprecipitation route without involvement of a surfactant. The powder X-ray diffraction method has been used to evaluate the crystalline nature and different phases consist of the formation of CuS NPs. Mainly hexagonal unit cells consist of the formation of CuS NP unit cells. Most of the surfaces are covered with rhombohedral microparticles with a smooth exterior and surface clustering, examined by SEM images, and the shape of NPs was spherical, having an average size of 23 nm, as confirmed by TEM analysis. This study has focused on the peroxidase-mimicking activity, superoxide dismutase (SOD)-mimicking activity, and chemosensor-based colorimetric determination and detection of epinephrine (EP) neurotransmitters with excellent selectivity. The CuS NPs catalyzed the oxidation of the oxidase substrate 3, 3-5, 5 tetramethyl benzidine (TMB) with the help of supplementary H2O2 that followed Michaelis-Menten kinetics with excellent Km and Vmax values calculated by the Lineweaver-Burk plot. Taking advantage of the drop in absorbance upon introduction of EP for the CuS NPs-TMB/H2O2 system, a colorimetric route has been developed for selective and real-time detection of EP. The sensitivity of the new colorimetric probe was vibrant, having a linear range of 0-16 µM, and achieved a low limit of detection of 457 nM. Moreover, the present nanosystem exhibited appreciable SOD-mimicking activity which could effectively remove O2•- from commercial cigarette smoke, along with it acting as a potential radical scavenger as well. The new nanosystem effectively scavenged •OH, O2.-, and metal chelation which were investigated calorimetrically.

Computational Screening for the Anticancer Potential of Seed-Derived Antioxidant Peptides: A Cheminformatic Approach.

Some seed-derived antioxidant peptides are known to regulate cellular modulators of ROS production, including those proposed to be promising targets of anticancer therapy. Nevertheless, research in this direction is relatively slow owing to the inevitable time-consuming nature of wet-lab experimentations. To help expedite such explorations, we performed structure-based virtual screening on seed-derived antioxidant peptides in the literature for anticancer potential. The ability of the peptides to interact with myeloperoxidase, xanthine oxidase, Keap1, and p47phox was examined. We generated a virtual library of 677 peptides based on a database and literature search. Screening for anticancer potential, non-toxicity, non-allergenicity, non-hemolyticity narrowed down the collection to five candidates. Molecular docking found LYSPH as the most promising in targeting myeloperoxidase, xanthine oxidase, and Keap1, whereas PSYLNTPLL was the best candidate to bind stably to key residues in p47phox. Stability of the four peptide-target complexes was supported by molecular dynamics simulation. LYSPH and PSYLNTPLL were predicted to have cell- and blood-brain barrier penetrating potential, although intolerant to gastrointestinal digestion. Computational alanine scanning found tyrosine residues in both peptides as crucial to stable binding to the targets. Overall, LYSPH and PSYLNTPLL are two potential anticancer peptides that deserve deeper exploration in future.

Peroxidase Mimetic Nanozymes in Cancer Phototherapy: Progress and Perspectives.

Nanomaterial-mediated cancer therapeutics is a fast developing field and has been utilized in potential clinical applications. However, most effective therapies, such as photodynamic therapy (PDT) and radio therapy (RT), are strongly oxygen-dependent, which hinders their practical applications. Later on, several strategies were developed to overcome tumor hypoxia, such as oxygen carrier nanomaterials and oxygen generated nanomaterials. Among these, oxygen species generation on nanozymes, especially catalase (CAT) mimetic nanozymes, convert endogenous hydrogen peroxide (H2O2) to oxygen (O2) and peroxidase (POD) mimetic nanozymes converts endogenous H2O2 to water (H2O) and reactive oxygen species (ROS) in a hypoxic tumor microenvironment is a fascinating approach. The present review provides a detailed examination of past, present and future perspectives of POD mimetic nanozymes for effective oxygen-dependent cancer phototherapeutics.

Effect of photodynamic treatments on quality and antioxidant properties of fresh-cut potatoes.

This study evaluated the feasibility of curcumin based photodynamic sterilization technology (PDT) applied to fresh-cut potato slices. Potato samples with 30 µmol L-1 curcumin solution were exposed to 420 nm light emitting diodes (LED) at a total dose of 0.7 kJ cm-2. Results showed that PDT inactivated 2.43 log CFU mL-1 of Escherichia coli (BL 21) and 3.18 log CFU mL-1 of Staphylococcus aureus and maintained the color, texture, weight as well as total solid content of treated potatoes. Additionally, loss of phenols and flavonoids was significantly prevented, increasing the total antioxidant capacity. This was attributed to changes in enzyme activity that PDT decreased the activity of polyphenol oxidase (PPO) and peroxidase (POD) by 59.7% and 47.8% and increased the activity of phenylalanine ammonia-lyase (PAL). Therefore, curcumin-based PDT has the potential to maintain the commercial quality of producing and achieving microbiological safety.

Polyphenol oxidase dominates the conversions of flavonol glycosides in tea leaves.

Flavonol glycosides are associated with astringency and bitterness of teas. To clarify the dominant enzymatic reaction of flavonol glycosides in tea leaves, the catalytic effects of polyphenol oxidase (PPO), peroxidase (POD) and ß-glucosidase were studied, with the maintaining rates of total flavonol glycosides (TFG) being 73.0%, 99.8% and 94.3%. PPO was selected for further investigations, including the effects of pH value (3.5 ~ 6.5), temperature (25 °C ~ 55 °C) and dosage (39 ~ 72 U/mL PPO and 36 U/mL PPO, 3 ~ 36 U/mL POD). The oxidation of flavonol glycosides were intensified at pH 6.5, with 51.8% and 15.4% of TFG maintained after PPO and PPO + POD treatments, suggesting an enhancement from POD. The sensitivity ranking to PPO was: myricetin glycosides > quercetin glycosides > kaempferol glycosides. The inhibitor treatment testified the leading role of PPO in catalyzing flavonol glycosides in tea leaves. Sugar moiety enhanced the docking affinity of flavonol glycosides for PPO. PPO shows the potential of modifying flavonol glycoside composition.

Influence of gelatin and collagen incorporation on peroxidase-mediated injectable pectin-based hydrogel and bioactivity of fibroblasts.

Pectin has recently attracted increasing attention for biomedical and pharmaceutical applications. Due to the lack of adhesion molecules in polysaccharides, phenolic hydroxyl conjugated gelatin was added to enzymatically-gellable peroxidase-modified pectin derivative and compared with phenolic hydroxyl -pectin/collagen. Both pectin and gelatin were modified by tyramine hydrochloride in the presence of EDC/NHS. The phenolic hydroxyl -pectin/phenolic hydroxyl -gelatin, phenolic hydroxyl-pectin/collagen, and phenolic hydroxyl -pectin hydrogels were prepared using horseradish peroxidase and hydrogen peroxide. The hydrogels were characterized by gelation time analysis. Morphology, enzymatic biodegradation, mechanical and swelling properties as well as water vapor transmission rate were also evaluated. Fibroblasts were cultured for 7 days, and the survival rate was evaluated using conventional MTT assay. Hydrogels composed of Ph-pectin/Ph-gelatin showed decreased biodegradation rate, and WVTR and further improved mechanical performance in comparison with other groups. Both phenolic hydroxyl -pectin/collagen and phenolic hydroxyl -pectin/phenolic hydroxyl -gelatin hydrogels exhibited porous structures. The hydrogels composed of collagen promoted cell survival rate 1.4 and 3.5 times compared to phenolic hydroxyl -gelatin and phenolic hydroxyl -pectin based hydrogels at the end of 7 days, respectively (p < 0.001). The study demonstrated the potential of enzymatically-gellable pectin-based hydrogels as cost-effective frameworks for use in tissue engineering applications.

The functionality of laccase- or peroxidase-treated potato flour: Role of interactions between protein and protein/starch.

Potato flour is used in bakery products, extruded products and snacks. However, it displays weaker gel strengths and thus the wholesome utility is curtailed significantly. To improve viscoelastic properties and stability of potato gels, herein potato flour was treated with laccase and peroxidase to create a protein network structure leading to stable gels. The results revealed that the secondary structure of potato proteins altered upon the enzyme treatment. The gels of peroxidase-treated potato flour (PPF) and laccase-treated potato flour (LPF) displayed larger anti-shear ability, thermal stability and stronger three-dimensional network structure compared to the native potato gel. The PPF and LPF gels exhibited stronger viscoelastic properties and structural stability compared to peroxidase-treated potato protein and laccase-treated potato protein gels. The outcome serves as a theoretical basis to improve the properties of potato gels and to promote the designing and the development of novel potato flour based functional food.

Abiotic stress elements in in vitro potato (Solanum tuberosum L.) exposed to air-based and liquid-based ultrasound: A comparative transcriptomic assessment.

Ultrasound (US) can modify the plant growth and development. Previous assessments of the transcriptome of in vitro potato (Solanum tuberosum L.) exposed to US transmitted through air (AB-US) or liquid (PE-US) revealed the up- or down-regulation of several stress-related differentially expressed genes (DEGs) related to abiotic stress. In a bid to better characterize stress-related elements over a four-week period, the transcriptome of AB-US was compared to that of PE-US. When comparing the controls of both treatments, DEGs related to hypoxia were not detected. Nevertheless, hypoxia-related DEGs were detected in the combination of liquid medium and ultrasonication. DEGs coding for chitinase, peroxidase, glutathione-S-transferase, transcription factors of ERF (ethylene responsive factor), DREB (dehydration-responsive element-binding), WRKY and MYB were also significantly highly expressed in PE-US, relative to AB-US. Up- and down-regulation of DEGs related to metabolic processes, and enzymes of the antioxidant system also confirm that PE-US is a more acute abiotic stress than AB-US. KEY MESSAGE: A transcriptomic analysis revealed that liquid-based ultrasonication was a stronger abiotic stressor than air-based ultrasonication. Of particular interest were the heat shock proteins and transcription factors in this comparison. Despite the ultrasound stress, explants survived and plantlets developed.

Intrinsic Peroxidase-Mimicking Ir Nanoplates for Nanozymatic Anticancer and Antibacterial Treatment.

The study of inorganic nanozymes to overcome the disadvantages of bio-enzymes, such as the requirement of optimized reaction conditions and lack of durability against environmental factors, is one of the most significant research topics at present. In this work, we comprehensively analyzed the intrinsic peroxidase-like activity of Ir-based nanoparticles, the biological and nanozymatic potentials of which have not yet been explored. These particles were synthesized by the galvanic replacement of Ag nanoplates with Ir. Through the confirmed peroxidase-like activity and hydrogen peroxide decomposition with free radical generation facilitated by these particles, the antibacterial and anticancer effects were successfully verified in vitro. The nanozyme-based therapeutic effect observed at concentrations at which these nanoparticles do not show cytotoxicity suggests that it is possible to achieve more precise and selective local treatment with these particles. The observed highly efficient peroxidase-like activity of these nanoparticles is attributed to the partially mixed composition of Ir-Ag-IrO2 formed through the galvanic replacement reaction in the synthetic process.

"Enzymatic and non-enzymatic antioxidant, anti-inflammatory and anticancer activities of Floscopa scandens Lour".

OBJECTIVE: To explore the total phenolic and flavonoid content, enzymatic, non-enzymatic antioxidant properties, anti-inflammation and anticancer activities of hexane, ethyl acetate and methanol extracts of Floscopa scandens (F. scandens). METHODS: Non-enzymatic antioxidant activity was examined by 2, 2-diphenyl-1-picrylhydrazyl assay, nitric oxide scavenging assay, hydroxyl radical scavenging assay, reducing power assay, hydrogen peroxide scavenging assay, superoxide scavenging assay and metal chelating assay. Enzymatic antioxidant ability was screened for the antioxidant enzymes such as ascorbate oxidase, peroxidase, catalase and polyphenol oxidase. The anti-inflammatory property was proved with the inhibition of protein denaturation and protease inhibitory assays. In vitro anticancer activity was assessed by cell viability assay. RESULTS: Methanol extract contained high amount of phenols (198.41 mg catechol equivalent/gram extract) and flavonoids (101.70 mg quercetin equivalent/gram extract) showed higher activity than hexane and ethyl acetate extracts in all experiments. Fresh plant showed considerable enzymatic antioxidant activity. CONCLUSION: The results revealed that the methanol extracts of F. scandens could be used as a potential source of antioxidant, anti-inflammatory and anticancer bioactive compounds.

A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity.

Alkene cleavage is a possibility to generate aldehydes with olfactory properties for the fragrance and flavor industry. A dye-decolorizing peroxidase (DyP) of the basidiomycete Pleurotus sapidus (PsaPOX) cleaved the aryl alkene trans-anethole. The PsaPOX was semi-purified from the mycelium via FPLC, and the corresponding gene was identified. The amino acid sequence as well as the predicted tertiary structure showed typical characteristics of DyPs as well as a non-canonical Mn2+-oxidation site on its surface. The gene was expressed in Komagataella pfaffii GS115 yielding activities up to 142 U/L using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. PsaPOX exhibited optima at pH 3.5 and 40 °C and showed highest peroxidase activity in the presence of 100 µM H2O2 and 25 mM Mn2+. PsaPOX lacked the typical activity of DyPs towards anthraquinone dyes, but oxidized Mn2+ to Mn3+. In addition, bleaching of ß-carotene and annatto was observed. Biotransformation experiments verified the alkene cleavage activity towards the aryl alkenes (E)-methyl isoeugenol, α-methylstyrene, and trans-anethole, which was increased almost twofold in the presence of Mn2+. The resultant aldehydes are olfactants used in the fragrance and flavor industry. PsaPOX is the first described DyP with alkene cleavage activity towards aryl alkenes and showed potential as biocatalyst for flavor production.

Function of hesperidin alleviating inflammation and oxidative stress responses in COPD mice might be related to SIRT1/PGC-1α/NF-κB signaling axis.

Purpose: Hesperidin has anti-inflammatory and anti-oxidant stress effects, but its functions in chronic obstructive pulmonary disease (COPD) remains unknown. This study analyzed the role of hesperidin in COPD mice, aiming to provide a basis for the hesperidin application.Materials and methods: Mice were injected with cigarette smoke extract (CSE) to construct COPD models and then treated with budesonide or hesperidin. Hematoxylin-eosin (HE) and TUNEL assays were used to observe the pathological changes and cell death of lung tissue. The levels of interleukin (IL)-6, IL-8, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) in bronchoalveolar lavage fluid (BLAF), as well as myeloperoxidase (MPO) content in lung tissues were confirmed. The expression levels of SIRT1, PGC-1α, and p65 proteins were measured by western blotting (WB) analysis.Results: CSE induced inflammatory cell infiltration and cell death in the lung tissues of mice, whereas budesonide and hesperidin effectively alleviated these pathological changes. The levels of IL-6, IL-8, and MDA in BLAF and pulmonary MPO content in the COPD mice were effectively increased, while the levels of SOD and CAT in BLAF were decreased, which could be reversed by budesonide and hesperidin. Moreover, the addition of budesonide or hesperidin reliably accelerated the expression levels of PGC-1α and SIRT1 but suppressed the phosphorylation of p65 in COPD mice. In general, high-dose hesperidin had a stronger regulatory effect on COPD mice.Conclusions: Hesperidin alleviated inflammation and oxidative stress responses in CES-induced COPD mice, associated with SIRT1/PGC-1α/NF-κB signaling axis, which might become a new direction for COPD treatment.

Virus-Like Fe3O4@Bi2S3 Nanozymes with Resistance-Free Apoptotic Hyperthermia-Augmented Nanozymitic Activity for Enhanced Synergetic Cancer Therapy.

Nanomaterials with intrinsic peroxidase-like activities are able to catalyze the oxidation of the substrate with the peroxide, which have been widely considered as artificial enzymatic agents in cancer therapy. However, current peroxidase catalytic oxidation treatments generating reactive oxygen species rely highly on hydrogen peroxide and pH, which limit greatly their therapeutic efficiency in the tumor microenvironment. Here, we report a strategy to construct the complex virus-like Fe3O4@Bi2S3 nanocatalysts (F-BS NCs) by connecting typical peroxidase Fe3O4 (MNPs) with a narrow band gap semiconductor Bi2S3 (BS) to enhance the enzymatic activity resorting to the limited intratumoral peroxide and efficient external photothermal stimuli. In this formulation, the integrated F-BS NCs induce cancer-cell death through mild photothermal treatment and sequential photothermal-stimulative catalysis of H2O2 into highly toxic •OH under 808 nm laser, which successfully realize a remarkable synergistic anticancer achievement.

Visualization and colorimetric determination of clenbuterol in pork by using magnetic beads modified with aptamer and complementary DNA as capture probes, and G-quadruplex/hemin and DNA antibody on the metal-organic framework MIL-101(Fe) acting as a peroxidase mimic.

A visualization strategy is described for the detection of clenbuterol (CLB). It is using of antibody against dsDNA and G-quadruplex/hemin labeled on a metal organic framework of type MIL-101(Fe) (G-quadruplex/hemin-anti-DNA/MIL-101) acting as a peroxidase mimetic, and magnetic beads modified with aptamer and complementary DNA (MB/Apt-cDNA) as capture probes. The detection reagent was prepared via the reactions between the double stranded DNA (Apt-cDNA) in capture probes and anti-DNA in peroxidase mimetic. In the presence of CLB, the aptamer on the magnetic beads preferentially binds CLB, and the peroxidase mimetic is released to the supernatant after magnetic separation. The released peroxidase mimetic can catalyze the TMB/H2O2 chromogenic system under mild conditions. This leads to the development of a blue-green coloration whose absorbance is measured at 650 nm. The detection limit is as low as 34 fM of CLB. The method was applied to the determination of CLB in pork samples and gave results that were consistent with data obtained with an ELISA kit. Graphical abstract A visualization strategy is described for the detection of clenbuterol. The selectivity of detection system for clenbuterol is excellent compared with other interferents. The method was applied to the determination of CLB in pork samples.

Purification of peroxidase enzyme from radish species in fast and high yield with affinity chromatography technique.

In this study, an effective single step affinity method is presented for purifying plant peroxidase (POD) enzymes from radish species. This method make possible to purify the enzymes in high yield and purity. Briefly, 10 different 4-amino benzohydrazide derivatives were synthesized and identified as new competitive POD inhibitors. Then, these derivatives were coupled to Sepharose 4B-L-Tyrosine support matrix by diazotization to form the affinity gels. Purification factors were recorded as 54.8% yield - 665-fold, 33.8% yield - 613-fold, 22.7% yield - 595-fold, 34.4% yield - 781-fold, 40.9% yield - 282-fold for turnip (T-POD), black radish (BR-POD), daikon (D-POD), sweet radish (SR-POD) and kohlrabi radish, (KR-POD), respectively. It has also been shown that the affinity gels, which prepared using the 4-amino 3-bromo benzohydrazide and 4-amino 2-nitro benzohydrazide molecules, capable to purify all radish species POD enzymes in high purity and yield.

A novel strategy for evaluation of natural products acting on the myeloperoxidase/hypochlorous acid system by combining high-performance liquid chromatography-photodiode array detection-chemiluminescence and ultrafiltration-mass spectrometry techniques.

Myeloperoxidase and its product hypochlorous acid are endogenous substances that are involved in the pathogenesis of many inflammatory diseases. In the present study, a novel evaluation strategy has been developed to screen for myeloperoxidase inhibitors and hypochlorous acid scavengers in natural products. The evaluation strategy uses two methods: a multi-hyphenated high-performance liquid chromatography-photodiode array detection-chemiluminescence online method, and an ultrafiltration-high-performance liquid chromatography-mass spectrometry method. Both were used for screening hypochlorous acid scavengers and myeloperoxidase inhibitors. After optimization of operating conditions, including pH of mobile phase and concentrations of myeloperoxidase, hypochlorous acid and luminol, the evaluation strategy was applied to Gardenia jasminoides Ellis (Rubiaceae) extract. Gardenia jasminoides Ellis (Rubiaceae) extract was found to both scavenge hypochlorous acid and inhibit myeloperoxidase. These different bioactivities originate from iridoid glycosides and quinic acid derivatives (25 hypochlorous acid scavengers) and crocetin derivatives (4 myeloperoxidase inhibitors). In a mouse model of acute lung injury, pulmonary histopathology showed that different constituents of Gardenia jasminoides Ellis (Rubiaceae) extract could also attenuate lung injury. Although the screening strategy has two arms, it is still simple, rapid, and effective. The strategy can also potentially be used to discriminate different activities of different constituents contained in the same natural product.

Guar gum blended alginate/agarose hydrogel as a promising support for the entrapment of peroxidase: Stability and reusability studies for the treatment of textile effluent.

Ginger peroxidase (GP) was entrapped into the hydrogels of guar gum (GG)-alginate/agarose and these immobilized GP preparations were employed for the treatment of textile effluent. GG is a natural hydrophilic polysaccharide, the average size of which increases in its hydrated form that helps in retaining the enzyme inside the entrapping support. Therefore, the activity retention by alginate-guar gum (ANGG) and agarose-guar gum (AGG) was higher than that of alginate and agarose alone. ANGG-GP and AGG-GP were highly stable against various physical and chemical denaturants during the decolorization of textile effluent. As compared to free GP, both the immobilized preparations were more efficient in the decolorization of textile effluent in batch processes. After 10th repeated use in batch processes, ANGG-GP and AGG-GP was quite effective in removing up to 68% and 55% of the color from textile effluent, respectively. Continuous packed bed reactors containing ANGG-GP and AGG-GP were able to decolorize around 80% and 69% of the effluent color, respectively, even after 30 days of their continuous operation at room temperature (30 °C). Genotoxicity of textile effluent was significantly reduced after GP mediated decolorization.

Overcoming Reductant Interference in Peroxidase-Based Assays for Hydrogen Peroxide Quantification.

A problem commonly encountered when using peroxidase-based methods for hydrogen peroxide quantification in biobased matrixes is interference due to the presence of endogenous reductants. Such assays are typically based on the generation of an oxidized reporter molecule in direct proportion to the amount of hydrogen peroxide reduced in the peroxidase-catalyzed reaction. Endogenous reductants confound such assays by reducing the oxidized reporter molecule, thus resulting in underestimates of hydrogen peroxide content. In the present work, we demonstrate how this problem can be circumvented by selectively oxidizing offending compounds by treatment with the oxidized reporter molecule prior to initiating the peroxidase reaction for hydrogen peroxide quantification. The approach is demonstrated using horseradish peroxidase, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), as the reporter molecule and a representative garlic paste as the hydrogen peroxide-containing biobased matrix. The approach is expected to be generally applicable to a wide range of peroxidase-based assays when applied to complex biobased systems.

Structural and functional study of a simple, rapid, and label-free DNAzyme-based DNA biosensor for optimization activity.

Peroxidase-mimicking DNAzyme has a potential to self-assemble into a G-quadruplex and shows peroxidase activity. In comparison to proteins, peroxidase-mimicking DNAzyme is less expensive and more stable. Herein, it is used in fabricating non-labeling biosensors. This paper investigates the structural and functional properties of a DNA biosensor based on split DNAzyme with a detection limit in nM range (9.48 nM). Two halves of DNAzyme were linked by a complementary sequence of DNA target. Hybridization of the DNA target pulled two DNAzyme halves apart and peroxidase activity decreased. This study can be divided into 3 stages. First, the characteristics of DNAzyme were studied by Circular Dichroism technique and UV-Vis spectroscopy to find out DNAzyme's optimum activity. It is worth to note that some divalent cations were used to form G-quadruplex, in addition to common monovalent cations. Furthermore, the hemin incubation was also optimized. Secondly, the structural and functional properties of two types of split DNAzyme were compared with DNAzyme. Thirdly, the hybridization of DNA target was monitored. The results revealed that peroxidase activities of split types decreased by half without any specific conformational changes. Interestingly, the catalytic activities of split DNAzymes could be promoted by adding Mg2+ . Besides, it was demonstrated that the structure, peroxidation reaction, and DNA target hybridization of 2:2 and 3:1 split modes were almost alike. It was also illustrated that magnesium promoted the possibility of hybridization.