Activity Of Horseradish Peroxidase Research Articles

Sol-Gel Films Doped with Enzymes and Banana Crude Extract as Sensing Materials for Spectrophotometric Determination.

Chromogenic enzymatic reactions are very convenient for the determination of various biochemically active compounds. Sol-gel films are a promising platform for biosensor development. The creation of sol-gel films with immobilized enzymes deserves attention as an effective way to create optical biosensors. In the present work, the conditions are selected to obtain sol-gel films doped with horseradish peroxidase (HRP), mushroom tyrosinase (MT) and crude banana extract (BE), inside the polystyrene spectrophotometric cuvettes. Two procedures are proposed: the use of tetraethoxysilane-phenyltriethoxysilane (TEOS-PhTEOS) mixture as precursor, as well as the use of silicon polyethylene glycol (SPG).In both types of films, the enzymatic activity of HRP, MT, and BE is preserved. Based on the kinetics study of enzymatic reactions catalyzed by sol-gel films doped with HRP, MT, and BE, we found that encapsulation in the TEOS-PhTEOS films affects the enzymatic activity to a lesser extent compared to encapsulation in SPG films. Immobilization affects BE significantly less than MT and HRP. The Michaelis constant for BE encapsulated in TEOS-PhTEOS films almost does not differ from the Michaelis constant for a non-immobilized BE. The proposed sol-gel films allow determining hydrogen peroxide in the range of 0.2-3.5 mM (HRP containing film in the presence of TMB), and caffeic acid in the ranges of 0.5-10.0 mM and 2.0-10.0 mM (MT- and BE-containing films, respectively). BE-containing films have been used to determine the total polyphenol content of coffee in caffeic acid equivalents; the results of the analysis are in good agreement with the results obtained using an independent method of determination. These films are highly stable and can be stored without the loss of activity for 2 months at +4 °C and 2 weeks at +25 °C.

Stepwise Optimization of Inducible Expression System for the Functional Secretion of Horseradish Peroxidase in Saccharomyces cerevisiae.

Horseradish peroxidase (HRP) is a plant-derived glycoprotein that can be developed as a food additive to cross-link proteins or biopolymers. Although Saccharomyces cerevisiae has advantages in the production of food-grade HRP, the low expressional level and inefficient secretion hindered its application values. After comparing the effects of constitutive and inducible expression on cell growth, the strength of HRP expression was roughly tuned by replacing core regions of the promoter in the GAL80-knockout strain and further finely tuned by terminator screening. Additionally, the most suitable signal peptide was selected, and the pre-peptide with pro-peptides was modified to balance the transport of HRP in the endoplasmic reticulum. The extracellular HRP activity of the best strain reached 13 506 U/L at the fermenter level, 330-fold higher than the previous result of 41 U/L in S. cerevisiae. The strategy can be applied to alleviate the inhibition of cell growth caused by the expression of toxic proteins and improve their secretion.

Nanozymes: Definition, Activity, and Mechanisms.

"Nanozyme" is used to describe various catalysts from immobilized inorganic metal complexes, immobilized enzymes to inorganic nanoparticles. Here, the history of nanozymes is dvescribed in detail, and they can be largely separated into two types. Type 1 nanozymes refer to immobilized catalysts or enzymes on nanomaterials, whichweredominant in the first decade since 2004. Type 2 nanozymes, which rely on the surface catalytic properties of inorganic nanomaterials, are the dominating type in the past decade. The definition of nanozymes is evolving, and a definition based on the same substrates and products as enzymes are able to cover most currently claimed nanozymes, although they may have different mechanisms compared to their enzyme counterparts. A broader definition can inspire application-based research to replace enzymes with nanomaterials for analytical, environmental, and biomedical applications. Comparison with enzymes also requires a clear definition of a nanozyme unit. Four ways of defining a nanozyme unit are described, with iron oxide and horseradish peroxidase activity comparison as examples in each definition. Growing work is devoted to understanding thecatalytic mechanism of nanozymes, which provides a basis for further rational engineering of active sites. Finally, future perspective of the nanozyme field is discussed.

Inactivation of horseradish peroxidase with crotonic acid for reprobing of western blotting

Inactivation of horseradish peroxidase (HRP) treatment is a conventional preference to stripping for sequential detections of different proteins of chemiluminescent western blotting (WB). However, little evidence exists on whether other chemical substances treatment can affects the biological activity of HRP during stripping and re-probing of WB blots. Here, we successfully develop 20% crotonic acid (CA) as an alternative to stripping to inhibit HRP used for sequential chemiluminescent WB on polyvinylidene difluoride (PVDF) and Nitrocellulose (NC) membrane. Moreover, NC blots incubation in CA (40 °C, 30min) allow us to perform three round HRP inhibition in sequential detections without losing transferred proteins and damaging membrane. Hence, the method will help us save time and valuable samples without the need to rerun gels.

Performance of a Flow-Through Enzyme Reactor Prepared from a Silica Monolith and an α-Poly(D-Lysine)-Enzyme Conjugate.

Horseradish peroxidase (HRP) is covalently bound in aqueous solution to polycationic α-poly(D-lysine) chains of ≈1000 repeating units length, PDL, via a bis-aryl hydrazone bond (BAH). Under the experimental conditions used, about 15 HRP molecules are bound along the PDL chain. The purified PDL-BAH-HRP conjugate is very stable when stored at micromolar HRP concentration in a pH 7.2 phosphate buffer solution at 4°C. When a defined volume of such a conjugate solution of desired HRP concentration (i.e., HRP activity) is added to a macro- and mesoporous silica monolith with pore sizes of 20-30µm as well as below 30nm, quantitative and stable noncovalent conjugate immobilization is achieved. The HRP-containing monolith can be used as flow-through enzyme reactor for bioanalytical applications at neutral or slightly alkaline pH, as demonstrated for the determination of hydrogen peroxide in diluted honey. The conjugate can be detached from the monolith by simple enzyme reactor washing with an aqueous solution of pH 5.0, enabling reloading with fresh conjugate solution at pH 7.2. Compared to previously investigated polycationic dendronized polymer-enzyme conjugates with approximately the same average polymer chain length, the PDL-BAH-HRP conjugate appears to be equally suitable for HRP immobilization on silica surfaces.

Improving the activity of horseradish peroxidase in betaine-based natural deep eutectic systems

In this work, the activity and thermostability of horseradish peroxidase (HRP) in betaine-based natural deep eutectic systems (NADESs) was measured and studied by molecular simulations.

Microfluidic technology for macro systems: Removal of textile dyes from wastewater in a microreactor

Wastewater from textile industry contains considerable amount of dissolved dye that can trigger environmental issues if is not treated properly. Numerous methods have been developed to degrade recalcitrant pollutants safely and utterly. Among them, enzymatic treatment of wastewater is gaining attention due to the enzyme’s specificity, easier manipulation, and generation of less toxic by-products. Still, the cost of enzymatic systems is the main limitation keeping the biocatalysts at lab-scale. Alternative solution for reducing the cost of enzymatic reaction systems is the use of microfluidic systems, which contribute to better mixing, process intensification and cleaner production. In this study, implementation of horseradish peroxidase for removal of the textile dye Acid Violet 109 is performed in a microfluidic reactor. The microreactor consists of three plunger pump units, two mixers and PTFE tube. The process parameters: residence time, dye, hydrogen peroxide, enzyme activity, the reactors’ diameter and length were optimized. Under the optimal process conditions: 30 mg/L dye concentration, 0.8 U/mL horseradish peroxidase activity, 0.1 mM hydrogen peroxide, 0.25 mm reactor’s diameter, 97,3 % removal was achieved at residence time of 6 min. The results from this study show that enzymatic microfluidic reactors are a convenient technology for dye removal.

Metal-organic framework (MOF) hybridized gold nanoparticles as a bifunctional nanozyme for glucose sensing.

Inspired by natural enzymes that possess multiple catalytic activities, here we develop a bifunctional metal-organic frame-work (MOF) for biosensing applications. Ultrasmall gold nano-particles (AuNPs) are grown in the internal cavities of an iron (Fe) porphyrin-based MOF to produce a hybridized nanozyme, AuNPs@PCN-224(Fe), in which AuNPs and PCN-224(Fe) exhibit the catalytic activity of glucose oxidase (GOx) and horseradish peroxidase (HRP), respectively. We established that the bifunctional nanozyme was capable of a cascade reaction to generate hydrogen peroxide in the presence of d-glucose and oxygen in situ, and subsequently activate a colorimetric or chemiluminescent substrate through HRP-mimicking catalytic activity. The nanozyme was selective over a range of other saccharides, and 93% of the catalytic activity was retained after being recycled five times.

Ferric heme b in aqueous micellar and vesicular systems: state-of-the-art and challenges.

Ferric heme b (= ferric protoporphyrin IX = hemin) is an important prosthetic group of different types of enzymes, including the intensively investigated and widely applied horseradish peroxidase (HRP). In HRP, hemin is present in monomeric form in a hydrophobic pocket containing among other amino acid side chains the two imidazoyl groups of His170 and His42. Both amino acids are important for the peroxidase activity of HRP as an axial ligand of hemin (proximal His170) and as an acid/base catalyst (distal His42). A key feature of the peroxidase mechanism of HRP is the initial formation of compound I under heterolytic cleavage of added hydrogen peroxide as a terminal oxidant. Investigations of free hemin dispersed in aqueous solution showed that different types of hemin dimers can form, depending on the experimental conditions, possibly resulting in hemin crystallization. Although it has been recognized already in the 1970s that hemin aggregation can be prevented in aqueous solution by using micelle-forming amphiphiles, it remains a challenge to prepare hemin-containing micellar and vesicular systems with peroxidase-like activities. Such systems are of interest as cheap HRP-mimicking catalysts for analytical and synthetic applications. Some of the key concepts on which research in this fascinating and interdisciplinary field is based are summarized, along with major accomplishments and possible directions for further improvement. A systematic analysis of the physico-chemical properties of hemin in aqueous micellar solutions and vesicular dispersions must be combined with a reliable evaluation of its catalytic activity. Future studies should show how well the molecular complexity around hemin in HRP can be mimicked by using micelles or vesicles. Because of the importance of heme b in virtually all biological systems and the fact that porphyrins and hemes can be obtained under potentially prebiotic conditions, ideas exist about the possible role of heme-containing micellar and vesicular systems in prebiotic times.

A strategy for the accurate detection of glucose in human serum based on the IFE effect of up-transformed nanoparticles

Here, the inner filter effect (IFE) was applied to construct a “turn off” mode up-conversion fluorescence and colorimetric dual-reading detection platform consisting of the chromogenic substance p-phenylenediamine (PPD) and NaYF4:Yb/Er@NaYF4 core–shell up-conversion nanoparticles (UCNPs) for the detection of glucose and hydrogen peroxide (H2O2) with high sensitivity and selectivity. In this strategy, hydrogen peroxide is produced in the process of glucose oxidation by catalyzing glucose oxidase (GOx). PPD is oxidized by H2O2 under the action of horseradish peroxidase (HRP), resulting in the transformation into its oxidation product PPDox, whose absorption peak coincides with the emission peak of UCNP at 540 nm, which can significantly quench the fluorescence of UCNP. The quenching efficiency is related to PPDox production, so the relationship between the glucose content and the fluorescence changes of UCNP can be established to realize the sensitive detection of glucose. UCNPs modified by –COOH and the oxidation product of PPD were negatively charged, which increased the distance between donor and recipient due to electrostatic repulsion. It was not conducive to the occurrence of distance-dependent fluorescence resonance energy transfer (FRET), which was also verified by fluorescence lifetime test. Under the optimal experimental conditions, the linear ranges of hydrogen peroxide and glucose were 2.5 ∼ 50 and 5 ∼ 80 μmol/L with the detection limit (LOD) of 0.32 and 0.83 μmol/L, respectively, which were lower than of some reported approaches. Furthermore, the system could be applied to the detection of glucose in actual serum sample and the results were consistency of commercial glucose meters which indicated that the protocol was reliable and potential applications for clinical diagnosis.

Development of Targeted Protein-Displaying Technology with a Novel Carbon Material

This study reports a new carbon material and its specific display of targeted protein. The properties of the carbon materials fabricated with carbon black MOGUL® were analyzed. The carbon materials were spherical structures with 55.421 µm as a median value. The specific surface area, pore volume, average pore diameter, and total of the acidic functional group were 130 m2·g-1, 0.55 cm3·g-1, 17.2 nm, and 0.29 mEq·g-1, respectively. The adsorption-desorption isoform of the carbon materials showed type IV of the hysteresis loop as defined by IUPAC, indicating non-uniform mesoporous structures (2-50 nm). The distribution of the log differential pore volume also indicated non-uniform porous structures because (i) the difference between the average pore size and the most frequent pore size was significant and (ii) the σ value was larger than the average value regarding the pore sizes. However, 10-90% of the integrated values of the log differential pore volume were 57.4% of the total integrated values, and the distribution was similar to the Gauss distribution model. Although the value of the total of the acidic functional group was 2.5-5.4 times lower than the values of the HPLC columns, the carbon materials require good scaffold quality rather than good HPLC quality. Therefore, the amounts could be enough for the scaffold of biotin hydrazide. To demonstrate the property of displaying the targeted proteins, carbon materials displaying biotin hydrazide by covalent bonding were prepared and avidin-labeled horse radish peroxidase (HRP) was bound to the biotin region. The carbon materials were porous structures, so the unspecific adsorption of HRP was estimated. Then, the maintenance ratios of HRP activities were analyzed in the repeated-use-with-wash processes after each evaluation, resulting in the activities of HRP on the carbon materials being treated with biotin hydrazide being significantly maintained compared to that of the ones without biotin hydrazide. The study revealed the properties of the carbon materials and indicated the display of HRP, suggesting that the carbon materials could be a new material for displaying targeted proteins.

The Formation of Protein Corona by Nanoplastics and Horseradish Peroxidase.

In theory, nanoplastics (NPs) can adsorb biological macromolecules, such as proteins, in the surrounding environment to form protein corona (PC). In this study, we focus on amino polystyrene (PS) NPs and horseradish peroxidase (HRP) to explore the dynamic process of the formation of PS-HRP PC and their influence on PS and HRP. This work used atomic force microscopy, laser particle size and Zeta potential analyzer, and UV-vis spectrophotometer. According to the adsorption behavior of HRP to NPs, the surface morphology characteristics of NPs can be observed to change at 60 min. Meanwhile, the increase in size and hydrodynamic diameter, the decrease in Zeta potential, surface roughness and HRP activity, and the change in HRP structure attest to the PC formation. The thickness of the PC was approximately 30 nm and there are differences in the dynamic and static variations in the size of the PC. The PC formation process progresses gradually from 0 min to 240 min. Overall, the formation of PS-HRP PC is identified, and the changes in its properties are confirmed from the perspective of nanoplastics and peroxidase, which help study the effects of nanoplastics on the environment and creatures.

Immobilization of horseradish peroxidase on hierarchically porous magnetic metal-organic frameworks for visual detection and efficient degradation of 2,4-dichlorophenol in simulated wastewater

Immobilization of enzymes onto solid carrier is an important way to improve the stability and reusability of enzymes. However, the enzymatic activity and catalytic efficiency are severely hampered due to enzyme leaching and increased substrate mass transfer resistance. Herein, for the first time, we reported a novel strategy of co-regionalization for enzyme immobilization using hierarchically porous magnetic metal-organic frameworks (HP-Zr-MOF@Fe3O4). The HP-Zr-MOF@Fe3O4 integrate magnetic characteristics and hierarchical porous structure for supporting horseradish peroxidase (HRP) via metal-ion affinity interaction, in which the mesoporous pores are suitable for entrapping enzyme while the microporous can be used to concentrate substrate to reduce the mass transfer resistance. Significantly, the prepared HRP@HP-Zr-MOF@Fe3O4 can efficiently suppress enzyme leaching from the HP-Zr-MOF@Fe3O4 skeleton and remain superior recovered activity (96.8 %) of the initial activity of free HRP, which exhibited excellent stability and reusability. Interestingly, the HRP@HP-Zr-MOF@Fe3O4 was used efficiently in the colorimetric detection and catalytic degradation of 2,4-dichlorophenol in simulated wastewater. The excellent property of HRP@HP-Zr-MOF@Fe3O4 was investigated through the analyses of kinetics and thermodynamics, demonstrating that the HRP@HP-Zr-MOF@Fe3O4 had significant specificity and binding affinity for the substrate compared with free HRP.

Effect of a Conical Cellulose Structure on Horseradish Peroxidase Biomacromolecules

The effect of a dielectric conical structure on the adsorption properties of an enzyme on mica was studied by atomic force microscopy (AFM) with the example of horseradish peroxidase (HRP). The cone used was a cellulose cone with a 60° apex angle. Namely, AFM allowed us to reveal an increase in the enzyme’s aggregation during its adsorption onto mica from the solution incubated near the cone apex for 40 min—as compared with the control enzyme samples incubated far away from the cone. In contrast, no change in the HRP adsorption properties was observed after shorter (10 min) incubation of the sample near the cone. The enzymatic activity of HRP was found to be the same for all the enzyme samples studied. Our findings should be considered upon designing biosensors (in particular, those intended for highly sensitive diagnostic applications) and bioreactors containing conical structural elements. Furthermore, since HRP is widely employed as a model enzyme in studies of external impacts on enzymes determining food quality, our data can be of use in the development of food-processing methods based on the use of electromagnetic radiation (microwave treatment, radiofrequency heating, etc.).

AFM Investigation of the Influence of Steam Flow through a Conical Coil Heat Exchanger on Enzyme Properties.

The present study is aimed at the revelation of subtle effects of steam flow through a conical coil heat exchanger on an enzyme, incubated near the heat exchanger, at the nanoscale. For this purpose, atomic force microscopy (AFM) has been employed. In our experiments, horseradish peroxidase (HRP) was used as a model enzyme. HRP is extensively employed as a model in food science in order to determine the influence of electromagnetic fields on enzymes. Adsorption properties of HRP on mica have been studied by AFM at the level of individual enzyme macromolecules, while the enzymatic activity of HRP has been studied by spectrophotometry. The solution of HRP was incubated either near the top or at the side of the conically wound aluminium pipe, through which steam flow passed. Our AFM data indicated an increase in the enzyme aggregation on mica after its incubation at either of the two points near the heat exchanger. At the same time, in the spectrophotometry experiments, a slight change in the shape of the curves, reflecting the HRP-catalyzed kinetics of ABTS oxidation by hydrogen peroxide, has also been observed after the incubation of the enzyme solution near the heat exchanger. These effects on the enzyme adsorption and kinetics can be explained by alterations in the enzyme hydration caused by the influence of the electromagnetic field, induced triboelectrically by the flow of steam through the heat exchanger. Our findings should thus be considered in the development of equipment involving conical heat exchangers, intended for either research or industrial use (including miniaturized bioreactors and biosensors). The increased aggregation of the HRP enzyme, observed after its incubation near the heat exchanger, should also be taken into account in analysis of possible adverse effects from steam-heated industrial equipment on the human body.

Synthetic Protocell as Efficient Bioreactor: Enzymatic Superactivity and Ultrasensitive Glucose Sensing in Urine.

It is believed that membraneless cellular condensates play a critical role in accelerating various slow and thermodynamically unfavorable biochemical processes. However, the exact mechanisms behind the enhanced activity within biocondensates remain poorly understood. Here, we report the fabrication of a high-performance integrated cascade bioplatform based on synthetic droplets for ultrasensitive glucose sensing. Using a horseradish peroxidase (HRP) and glucose oxidase (GOx) cascade pair, we report an unprecedented enhancement in the catalytic activity of HRP inside the synthetic membraneless droplet. Liquidlike membraneless droplets have been prepared via multivalent electrostatic interactions between adenosine triphosphate (ATP) and poly(diallyldimethylammonium chloride) (PDADMAC) in an aqueous medium. Compartmentalized enzymes (GOx/HRP@Droplet) exhibit high encapsulation efficiency, low leakage, prolong retention of activity, and exceptional stability toward protease digestion. Using an HRP@Droplet composite, we have shown that the enzymatic reaction within the droplet follows the classical Michaelis-Menten model. Our findings reveal remarkable enhancement in the catalytic activity of up to 100- and 51-fold for HRP@Droplet and GOx/HRP@Droplet, respectively. These enhanced activities have been explained on the basis of increased local concentrations of enzymes and substrates, along with altered conformations of sequestered enzymes. Furthermore, we have utilized highly efficient and recyclable GOx/HRP@Droplet composite to demonstrate ultrasensitive glucose sensing with a limit of detection of 228 nM. Finally, the composite platform has been exploited to detect glucose in spiked urine samples in solution and filter paper. Our present study illustrates the unprecedented activity of the compartmentalized enzymes and paves the way for next-generation composite bioreactors for a wide range of applications.

The Effect of a Rotating Cone on Horseradish Peroxidase Aggregation on Mica Revealed by Atomic Force Microscopy.

Our study reported herein aims to determine whether an electromagnetic field, induced triboelectrically by a metallic cone, rotating at a frequency of 167 Hz, has an effect on the properties of the horseradish peroxidase (HRP) enzyme. Atomic force microscopy (AFM) was employed to detect even the most subtle effects on single enzyme molecules. In parallel, a macroscopic method (spectrophotometry) was used to reveal whether the enzymatic activity of HRP in solution was affected. An aqueous solution of the enzyme was incubated at a distance of 2 cm from the rotating cone. The experiments were performed at various incubation times. The control experiments were performed with a non-rotating cone. The incubation of the HRP solution was found to cause the disaggregation of the enzyme. At longer incubation times, this disaggregation was found to be accompanied by the formation of higher-order aggregates; however, no change in the HRP enzymatic activity was observed. The results of our experiments could be of interest in the development of enzyme-based biosensors with rotating elements such as stirrers. Additionally, the results obtained herein are important for the correct interpretation of data obtained with such biosensors.

Atomic Force Microscopy Study of the Effect of an Electric Field, Applied to a Pyramidal Structure, on Enzyme Biomolecules.

The influence of an external constant strong electric field, formed using a pyramidal structure under a high electric potential, on an enzyme located near its apex, is studied. Horseradish peroxidase (HRP) is used as a model. In our experiments, a 27 kV direct current (DC) voltage was applied to two electrodes with a conducting pyramidal structure attached to one of them. The enzyme particles were visualized by atomic force microscopy (AFM) after the adsorption of the enzyme from its 0.1 µM solution onto mica AFM substrates. It is demonstrated that after the 40 min exposure to the electric field, the enzyme forms extended structures on mica, while in control experiments compact HRP particles are observed. After the exposure to the electric field, the majority of mica-adsorbed HRP particles had a height of 1.2 nm (as opposed to 1.0 nm in the case of control experiments), and the contribution of higher (>2.0 nm) particles was also considerable. This indicates the formation of high-order HRP aggregates under the influence of an applied electric field. At that, the enzymatic activity of HRP against its substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) remains unaffected. These results are important for studying macroscopic effects of strong electromagnetic fields on enzymes, as well as for the development of cellular structure models.

A hydrogel-based mechanical metamaterial for the interferometric profiling of extracellular vesicles in patient samples.

The utility of mechanical metamaterials for biomedical applications has seldom been explored. Here we show that a metamaterial that is mechanically responsive to antibody-mediated biorecognition can serve as an optical interferometric mask to molecularly profile extracellular vesicles in ascites fluid from patients with cancer. The metamaterial consists of a hydrogel responsive to temperature and redox activity functionalized with antibodies to surface biomarkers on extracellular vesicles, and is patterned into micrometric squares on a gold-coated glass substrate. Through plasmonic heating, the metamaterial is maintained in a transition state between a relaxed form and a buckled state. Binding of extracellular vesicles from the patient samples to the antibodies on the hydrogel causes it to undergo crosslinking, induced by free radicals generated via the activity of horseradish peroxidase conjugated to the antibodies. Hydrogel crosslinking causes the metamaterial to undergo fast chiral re-organization, inducing amplified changes in its mechanical deformation and diffraction patterns, which are detectable by a smartphone camera. The mechanical metamaterial may find broad utility in the sensitive optical immunodetection of biomolecules.

Oxygen-terminated few-layered Ti3C2Tx MXene nanosheets as peroxidase-mimic nanozyme for colorimetric detection of kanamycin

Nanomaterials-based bioinspired enzyme mimics are gaining increased attention as alternatives to biocatalysts. Herein, we report synthesizing oxygen-terminated few-layered titanium-based MXene nanosheets (OFL-Ti-MN). OFL-Ti-MN possesses horseradish peroxidase (HRP) activity in catalyzing the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2), which turns the solution color bluish-green. The solution color fades quickly when kanamycin (KAN) is added to this system. This reaction indicates that KAN can prevent color change in the OFL-Ti-MN/TMB-H2O2 system. Based on this strategy, we developed an OFL-Ti-MN-based colorimetric sensor to detect and quantify KAN. The sensor exhibited a dynamic range from 15.28 nM to 46.14 μM and a calculated limit of detection (LOD) of 15.28 nM. From the insight gained from the peroxidase-mimic property of OFL-Ti-MN, we proposed a mechanism for the inhibition effect of KAN on peroxidase and peroxidase-mimic enzymes. The proposed mechanism can potentially help elucidate the reasons for the antibacterial function of KAN and its side effects in humans.