3,3′,5,5′-Tetramethylbenzidine Can Be Unsuitable for Identifying Peroxidases

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Learn More CiteCitationCitation and abstractCitation and referencesMore citation options ShareShare onFacebookX (Twitter)WeChatLinkedInRedditEmailJump toExpandCollapse EditorialAugust 23, 20243,3′,5,5′-Tetramethylbenzidine Can Be Unsuitable for Identifying PeroxidasesClick to copy article linkArticle link copied!Chao Wang*Chao WangCollege of Pharmacy, Oregon State University, 2730 South Moody Avenue, Portland, Oregon 97201 United States*Email: [email protected]More by Chao Wanghttps://orcid.org/0000-0003-3137-1632Open PDFACS SensorsCite this: ACS Sens. 2024, 9, 8, 3808–3809Click to copy citationCitation copied!https://pubs.acs.org/doi/10.1021/acssensors.4c01911https://doi.org/10.1021/acssensors.4c01911Published August 23, 2024 Publication History Received 26 July 2024Published online 23 August 2024Published in issue 23 August 2024editorialCopyright © Published 2024 by American Chemical Society. This publication is available under these Terms of Use. Request reuse permissionsThis publication is licensed for personal use by The American Chemical Society. ACS PublicationsCopyright © Published 2024 by American Chemical SocietySubjectswhat are subjectsArticle subjects are automatically applied from the ACS Subject Taxonomy and describe the scientific concepts and themes of the article.AssaysOxidation reactionsPeptides and proteinsReagentsRedox reactionsSince scientists began studying peroxidases (e.g., horseradish peroxidase, HRP), 3,3′,5,5′-tetramethylbenzidine (TMB) has become the most commonly used chromogenic substrate, with a history of over 50 years. (1) Its chromogenic reaction is based on the catalytic oxidation of TMB by HRP in the presence of H2O2, converting it from a reduced state to an oxidized state (oxTMB), and it changes from transparent to blue and finally yellow. (2) This allows for qualitative or quantitative determination of reaction states and activities through direct observation or spectrophotometry.Scientists' choice of TMB was not arbitrary but rational. Compared to other colorimetric reagents such as o-phenylenediamine (OPD) or 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), TMB is more sensitive due to its higher molar absorption coefficient (ε652 nm = 39000 M–1 cm–1), surpassing OPD (ε417 nm = 16700 M–1 cm–1) and ABTS (ε420 nm = 36000 M–1 cm–1). (3) Furthermore, compared with its derivatives, TMB exhibits the lowest potential carcinogenicity, higher sensitivity, better color purity of the colorimetric product, and reasonable stability of the oxidation product. (4) These factors cement TMB's status as the "chosen substrate" in peroxidase colorimetric assays.Despite these advantages and widespread application in peroxidase or peroxidase-like enzyme detection, TMB suffers from a significant flaw: its lack of specificity. For instance, in another common catalytic oxidation reaction, the Fenton reaction, TMB is also frequently used as a chromogenic reagent to indicate Fenton reagent activity. (5) In both pathways─peroxidase catalysis and Fenton reaction─TMB is oxidized to oxTMB in the presence of H2O2, but the reaction mechanisms differ. In peroxidase reactions, the enzyme catalyzes the two-electron oxidation of TMB by H2O2, whereas in the Fenton reaction, Fenton reagents (such as Fe2+ or Cu1+) react with H2O2 to produce highly oxidative hydroxyl radicals (•OH), which subsequently oxidize TMB to oxTMB. (6) Researchers often conflate these two reactions, considering any reagent or nanoparticle that changes TMB color in the presence of H2O2 as a peroxidase-like enzyme or nanozyme, (7) thereby confusing the research field and readers.Moreover, the situation is even worse. Besides the chromogenic reaction in systems containing H2O2 (regardless of whether it is a peroxidase catalysis or a Fenton reaction), TMB can also easily be oxidized in many common redox reaction systems, such as solutions containing Fe3+ or Cu2+, but lacking H2O2. (8) This further distances TMB from the title of "specific peroxidase reagent" (Scheme 1). Chemically, TMB has a low redox potential (0.186 V), (4) indicating its reducing properties, making it susceptible not only to strong oxidants (such as •OH, 2.81 V) (9) but also to moderately oxidizing agents (such as Fe3+, 0.77 V; Cu2+, 0.34 V), (10) or even prolonged exposure to light.Scheme 1Scheme 1. TMB Can Be Oxidized into oxTMB through Multiple PathwaysHigh Resolution ImageDownload MS PowerPoint SlideTherefore, TMB is not truly a specific assay reagent for peroxidase or Fenton reaction but rather a broad-spectrum chromogenic reagent for oxidative reactions.In conclusion, when the type of reactant being tested is known, TMB is suitable to determine the reaction activity and kinetics of peroxidase or Fenton reactions. However, prematurely attributing a reagent's involvement in a reaction system solely based on the oxidation and chromogenic reaction of TMB can lead to misunderstanding, with further investigation into the mechanism being crucial to determining the exact nature of the reagent involved. As mentioned above, Fe3+ and Cu2+ alone can oxidize TMB, and we cannot assume these metal ions are peroxidase or peroxidase-like enzymes. Using more specific assay reagents with defined reaction pathways is essential to accurately identify the type of reagent involved in the reaction (6)─if you truly want to know.Author InformationClick to copy section linkSection link copied!Corresponding AuthorChao Wang, College of Pharmacy, Oregon State University, 2730 South Moody Avenue, Portland, Oregon 97201 United States, https://orcid.org/0000-0003-3137-1632, Email: [email protected]NotesViews expressed in this editorial are those of the author and not necessarily the views of the ACS.ReferencesClick to copy section linkSection link copied! This article references 10 other publications. 1Engvall, E.; Perlmann, P. Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry 1971, 8 (9), 871– 874, DOI: 10.1016/0019-2791(71)90454-X Google Scholar1Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin GEngvall, Eva; Perlmann, PeterImmunochemistry (1971), 8 (9), 871-4CODEN: IMCHAZ; ISSN:0019-2791. An immunochem. assay for antibody is described which parallels a radioactive method (L. Wide and J. Porath, 1966) except that the antigen std. is labeled with enzyme instead of radioisotope. The sensitivities of the 2 methods are comparable. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE38XpslOhuw%253D%253D&md5=911feb5cbd6e1f2db2a43bdf26ece7692Zhu, C.; Yang, H.; Cao, X.; Hong, Q.; Xu, Y.; Wang, K.; Shen, Y.; Liu, S.; Zhang, Y. Decoupling of the Confused Complex in Oxidation of 3,3′,5,5′-Tetramethylbenzidine for the Reliable Chromogenic Bioassay. Anal. Chem. 2023, 95 (44), 16407– 16417, DOI: 10.1021/acs.analchem.3c03998 Google ScholarThere is no corresponding record for this reference.3Jiang, B.; Duan, D.; Gao, L.; Zhou, M.; Fan, K.; Tang, Y.; Xi, J.; Bi, Y.; Tong, Z.; Gao, G. F. Standardized assays for determining the catalytic activity and kinetics of peroxidase-like nanozymes. Nat. Protoc. 2018, 13 (7), 1506– 1520, DOI: 10.1038/s41596-018-0001-1 Google Scholar3Standardized assays for determining the catalytic activity and kinetics of peroxidase-like nanozymesJiang, Bing; Duan, Demin; Gao, Lizeng; Zhou, Mengjie; Fan, Kelong; Tang, Yan; Xi, Juqun; Bi, Yuhai; Tong, Zhou; Gao, George Fu; Xie, Ni; Tang, Aifa; Nie, Guohui; Liang, Minmin; Yan, XiyunNature Protocols (2018), 13 (7), 1506-1520CODEN: NPARDW; ISSN:1750-2799. (Nature Research) Nanozymes are nanomaterials exhibiting intrinsic enzyme-like characteristics that have increasingly attracted attention, owing to their high catalytic activity, low cost and high stability. This combination of properties has enabled a broad spectrum of applications, ranging from biol. detection assays to disease diagnosis and biomedicine development. Since the intrinsic peroxidase activity of Fe3O4 nanoparticles (NPs) was first reported in 2007, >40 types of nanozymes have been reported that possess peroxidase-, oxidase-, haloperoxidase- or superoxide dismutase-like catalytic activities. Given the complex interdependence of the physicochem. properties and catalytic characteristics of nanozymes, it is important to establish a std. by which the catalytic activities and kinetics of various nanozymes can be quant. compared and that will benefit the development of nanozyme-based detection and diagnostic technologies. Here, we first present a protocol for measuring and defining the catalytic activity units and kinetics for peroxidase nanozymes, the most widely used type of nanozyme. In addn., we describe the detailed exptl. procedures for a typical nanozyme strip-based biol. detection test and demonstrate that nanozyme-based detection is repeatable and reliable when guided by the presented nanozyme catalytic std. The catalytic activity and kinetics assays for a nanozyme can be performed within 4 h. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlWjsb7E&md5=3ff2c0e19222bb1dd39f0bf3f1a367564Zhang, X.; Yang, Q.; Lang, Y.; Jiang, X.; Wu, P. Rationale of 3,3′,5,5′-Tetramethylbenzidine as the Chromogenic Substrate in Colorimetric Analysis. Anal. Chem. 2020, 92 (18), 12400– 12406, DOI: 10.1021/acs.analchem.0c02149 Google Scholar4Rationale of 3,3',5,5'-Tetramethylbenzidine as the Chromogenic Substrate in Colorimetric AnalysisZhang, Xiao; Yang, Qin; Lang, Yunhe; Jiang, Xia; Wu, PengAnalytical Chemistry (Washington, DC, United States) (2020), 92 (18), 12400-12406CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society) Horseradish peroxidase (HRP)-based assays feature particular interests because of the simple colorimetric readout. In these assays, 3,3',5,5'-tetramethylbenzidine (TMB) is the most widely used chromogenic substrates for HRP. The later research in nanozyme and DNAzyme also used TMB (the chosen substrate) because they are both HRP-mimics. It should be noted that the substrate of HRP is not just limited to TMB but, in fact, a broad range of benzidine derivs. However, except decreased carcinogenicity due to tetrasubstitution of benzidine, the rationale for the chosen substrate TMB is not clear yet. Here, we addressed such a fundamental issue from the chem. point of view. Nine benzidine derivs. featuring varied properties (different substitution groups and varied no. of substitutions) were selected and investigated with four typical TMB-involved chromogenic systems. Among the existing benzidine substrates that are used for peroxidase-based assays, TMB exhibited the highest sensitivity, better color purity of colored products, and reasonable stability of oxidn. products. Moreover, two tetrasubstituted benzidine derivs. other than TMB (4OCH3 and 2OCH32CH3) were synthesized for comparison. It turned out that the performances (sensitivity, color purity, and stability of the colored products) of TMB are still superior, thus chem. confirming its status of "the chosen substrate" in colorimetric assays. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsF2nsLjO&md5=ad835f23fdd4cfc29da6bec8f4e71b315Lai, W.; Wei, Q.; Zhuang, J.; Lu, M.; Tang, D. Fenton reaction-based colorimetric immunoassay for sensitive detection of brevetoxin B. Biosens. Bioelectron. 2016, 80, 249– 256, DOI: 10.1016/j.bios.2016.01.088 Google Scholar5Fenton reaction-based colorimetric immunoassay for sensitive detection of brevetoxin BLai, Wenqiang; Wei, Qiaohua; Zhuang, Junyang; Lu, Minghua; Tang, DianpingBiosensors & Bioelectronics (2016), 80 (), 249-256CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.) We designed a new colorimetric immunoassay for sensitive monitoring of brevetoxin B (BTB) using enzyme-controlled Fenton reaction with a high-resoln. 3,3',5,5'-tetramethylbenzidine (TMB)-based visual colored system. Upon addn. of hydrogen peroxide (H2O2), the equiv. iron(II) could be first converted into iron(III) and free hydroxyl radical (•OH) via the classical Fenton reaction. Then the as-produced iron(III) and •OH could cause a perceptible change from colorless to blue with the increasing H2O2 concn. in the presence of TMB. Based on Fenton reaction-triggered visual colored system, a novel competitive-type colorimetric enzyme immunoassay was developed for the quant. screening of target BTB on the bovine serum albumin-BTB-modified magnetic bead using glucose oxidase/anti-BTB antibody-labeled gold nanoparticle as the signal-transduction tag. Upon target BTB introduction, the analyte competed with the conjugated BTB on the magnetic bead for anti-BTB antibody on gold nanoparticle. The carried glucose oxidase with the gold nanoparticle could implement the oxidn. of glucose to produce H2O2, and the generated H2O2 promoted the above-mentioned Fenton reaction for color development. Under the optimal conditions, the absorbance decreased with the increasing target BTB in the range from 0.1 to 150 ng kg-1 with a low detection limit (LOD) of 0.076 ng kg-1. The LOD was 500-fold lower than that of commercialized Abraxis BTB ELISA kit. Non-specific adsorption was not obsd. The precision, reproducibility and specificity were acceptable. Finally, the method accuracy was also validated for monitoring spiked seafood samples, giving results well matched with the referenced brevetoxin ELISA kit. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xit1SgtLg%253D&md5=eed2c339abf42a041d37ef48c38357746Robert, A.; Meunier, B. How to Define a Nanozyme. ACS Nano 2022, 16 (5), 6956– 6959, DOI: 10.1021/acsnano.2c02966 Google Scholar6How to Define a NanozymeRobert, Anne; Meunier, BernardACS Nano (2022), 16 (5), 6956-6959CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society) A review. Over the past 15 years, many articles have considered "nanozymes" as ferromagnetic nanoparticles having an "intrinsic peroxidase-like activity" in the presence of hydrogen peroxide. However, the definition and the catalytic activity of these nanozymes have been questioned. The present Perspective reports the main criteria that are essential to classify a nanoparticle as a nanozyme. It is important to consider that not all nanoparticles able to generate hydroxyl radicals in the presence of hydrogen peroxide without catalytic activity can be registered as nanozymes. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xht1Sgs7vL&md5=457641db0f966c8feb07109766629b387Wei, H.; Gao, L.; Fan, K.; Liu, J.; He, J.; Qu, X.; Dong, S.; Wang, E.; Yan, X. Nanozymes: A clear definition with fuzzy edges. Nano Today 2021, 40, 101269, DOI: 10.1016/j.nantod.2021.101269 Google Scholar7Nanozymes: A clear definition with fuzzy edgesWei, Hui; Gao, Lizeng; Fan, Kelong; Liu, Juewen; He, Jiuyang; Qu, Xiaogang; Dong, Shaojun; Wang, Erkang; Yan, XiyunNano Today (2021), 40 (), 101269CODEN: NTAOCG; ISSN:1748-0132. (Elsevier Ltd.) Most nanozyme research has been driven by applications, where the simple goal is the replacement of natural enzymes with more stable, cost-effective and sometimes more active nanomaterials. While in such work, nanozymes can certainly be called catalytic nanomaterials, we believe the conceptualization of these research efforts was encouraged by the name of nanozyme. nanozymes with HRP-like activity have been widely used in ELISA. The nanozyme-based ELISA had a detection limit of 0.67 pg/mL, which is approx. 110-fold better than the HRP-based ELISA. Natural enzymes often suffer from a lack of stability, preventing applications in harsh environments such as sea water or gastric acid. Nanozymes on the other hand have shown excellent stability under such conditions. Vanadium pentoxide (V2O5) nanozymes can substitute vanadium haloperoxidases for antifouling applications. Benefiting from the high stability of V2O5 nanowires, the nanozymes were painted on a stainless steel plate fixed to a boat hull and demonstrated effective suppression of biofouling in seawater for up to 60 days. Studies have also shown that nanozymes fulfill the physiol. function of natural enzymes both on the cellular and animal levels. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvVKgurrK&md5=c9f1c526bf4e2751cd12d3ec22b57f538Zhang, L.; Du, J. Selective sensing of submicromolar iron(III) with 3,3′,5,5′-tetramethylbenzidine as a chromogenic probe. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 2016, 158, 24– 28, DOI: 10.1016/j.saa.2016.01.012 Google ScholarThere is no corresponding record for this reference.(a) Chen, L.; Li, Y.; Sun, P.; Chen, H.; Li, H.; Liu, J.; Chen, Z.; Wang, B. A Facile Colorimetric Method for Ultra-rapid and Sensitive Detection of Copper Ions in Water. Journal of Inorganic and Organometallic Polymers and Materials 2022, 32 (7), 2473– 2481, DOI: 10.1007/s10904-022-02255-7 Google ScholarThere is no corresponding record for this reference.9Koppenol, W. H.; Liebman, J. F. The oxidizing nature of the hydroxyl radical. A comparison with the ferryl ion (FeO2+). J. Phys. Chem. 1984, 88 (1), 99– 101, DOI: 10.1021/j150645a024 Google Scholar9The oxidizing nature of the hydroxyl radical. A comparison with the ferryl ion (FeO2+)Koppenol, W. H.; Liebman, Joel F.Journal of Physical Chemistry (1984), 88 (1), 99-101CODEN: JPCHAX; ISSN:0022-3654. For the std. redn. potential of the hydroxyl radical/hydroxide couple, 2 values are found in the literature, 2.0 and 1.4 V. Thermochem. data yield 1.98 V for E°(·OHg/OH-aq). Hydration of the hydroxyl radical by -5 kcal/mol changes this value to 1.77 V for E°'(·OHaq/OH-aq), which is still much higher than 1.4 V. It is concluded that the latter value is incorrect. The following thermodn. quantities are derived from, or consistent with, the new E°' value: ΔG°f(·OHaq) = +3.2 kcal/mol; E°'(·OHaq/H2OI) = 2.59 V at pH 0; ΔG°f(O-·aq) = 19.5 kcal/mol; E°'(O-·aq/OH-aq) = 1.64 V at pH 14; ΔG°f(O3-·aq) = 11.7 kcal/mol; E°(O3g/O3-·aq) = 1.19 V; and E°'(H2O2aq/·OHaq, H2OI) = 0.46 V at pH 7. The redn. of hydrogen peroxide by ferrous complexes might yield the ferryl ion (FeO2+-chelate). If the chelating agent is a porphyrin (compd. II) the redn. potential of the couple ferryl/ferriporphyrin is estd. to be 0.9 V. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXktFGlsw%253D%253D&md5=d64f4af2d14cc6cd20b0dee78f74028e10Bard, A. J.; Parsons, R.; Jordan, J. Standard Potentials in Aqueous Solution; Taylor & Francis, 1985. DOI: 10.1201/9780203738764 .Google ScholarThere is no corresponding record for this reference.Cited By Click to copy section linkSection link copied!This article has not yet been cited by other publications.Download PDFFiguresReferencesOpen PDF Get e-AlertsGet e-AlertsACS SensorsCite this: ACS Sens. 2024, 9, 8, 3808–3809Click to copy citationCitation copied!https://doi.org/10.1021/acssensors.4c01911Published August 23, 2024 Publication History Received 26 July 2024Published online 23 August 2024Published in issue 23 August 2024Copyright © Published 2024 by American Chemical Society. This publication is available under these Terms of Use. Request reuse permissionsArticle Views-Altmetric-Citations-Learn about these metrics closeArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.Recommended Articles FiguresReferencesScheme 1Scheme 1. TMB Can Be Oxidized into oxTMB through Multiple PathwaysHigh Resolution ImageDownload MS PowerPoint SlideReferences This article references 10 other publications. 1Engvall, E.; Perlmann, P. Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry 1971, 8 (9), 871– 874, DOI: 10.1016/0019-2791(71)90454-X 1Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin GEngvall, Eva; Perlmann, PeterImmunochemistry (1971), 8 (9), 871-4CODEN: IMCHAZ; ISSN:0019-2791. An immunochem. assay for antibody is described which parallels a radioactive method (L. Wide and J. Porath, 1966) except that the antigen std. is labeled with enzyme instead of radioisotope. The sensitivities of the 2 methods are comparable. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE38XpslOhuw%253D%253D&md5=911feb5cbd6e1f2db2a43bdf26ece7692Zhu, C.; Yang, H.; Cao, X.; Hong, Q.; Xu, Y.; Wang, K.; Shen, Y.; Liu, S.; Zhang, Y. Decoupling of the Confused Complex in Oxidation of 3,3′,5,5′-Tetramethylbenzidine for the Reliable Chromogenic Bioassay. Anal. Chem. 2023, 95 (44), 16407– 16417, DOI: 10.1021/acs.analchem.3c03998 There is no corresponding record for this reference.3Jiang, B.; Duan, D.; Gao, L.; Zhou, M.; Fan, K.; Tang, Y.; Xi, J.; Bi, Y.; Tong, Z.; Gao, G. F. Standardized assays for determining the catalytic activity and kinetics of peroxidase-like nanozymes. Nat. Protoc. 2018, 13 (7), 1506– 1520, DOI: 10.1038/s41596-018-0001-1 3Standardized assays for determining the catalytic activity and kinetics of peroxidase-like nanozymesJiang, Bing; Duan, Demin; Gao, Lizeng; Zhou, Mengjie; Fan, Kelong; Tang, Yan; Xi, Juqun; Bi, Yuhai; Tong, Zhou; Gao, George Fu; Xie, Ni; Tang, Aifa; Nie, Guohui; Liang, Minmin; Yan, XiyunNature Protocols (2018), 13 (7), 1506-1520CODEN: NPARDW; ISSN:1750-2799. (Nature Research) Nanozymes are nanomaterials exhibiting intrinsic enzyme-like characteristics that have increasingly attracted attention, owing to their high catalytic activity, low cost and high stability. This combination of properties has enabled a broad spectrum of applications, ranging from biol. detection assays to disease diagnosis and biomedicine development. Since the intrinsic peroxidase activity of Fe3O4 nanoparticles (NPs) was first reported in 2007, >40 types of nanozymes have been reported that possess peroxidase-, oxidase-, haloperoxidase- or superoxide dismutase-like catalytic activities. Given the complex interdependence of the physicochem. properties and catalytic characteristics of nanozymes, it is important to establish a std. by which the catalytic activities and kinetics of various nanozymes can be quant. compared and that will benefit the development of nanozyme-based detection and diagnostic technologies. Here, we first present a protocol for measuring and defining the catalytic activity units and kinetics for peroxidase nanozymes, the most widely used type of nanozyme. In addn., we describe the detailed exptl. procedures for a typical nanozyme strip-based biol. detection test and demonstrate that nanozyme-based detection is repeatable and reliable when guided by the presented nanozyme catalytic std. The catalytic activity and kinetics assays for a nanozyme can be performed within 4 h. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlWjsb7E&md5=3ff2c0e19222bb1dd39f0bf3f1a367564Zhang, X.; Yang, Q.; Lang, Y.; Jiang, X.; Wu, P. Rationale of 3,3′,5,5′-Tetramethylbenzidine as the Chromogenic Substrate in Colorimetric Analysis. Anal. Chem. 2020, 92 (18), 12400– 12406, DOI: 10.1021/acs.analchem.0c02149 4Rationale of 3,3',5,5'-Tetramethylbenzidine as the Chromogenic Substrate in Colorimetric AnalysisZhang, Xiao; Yang, Qin; Lang, Yunhe; Jiang, Xia; Wu, PengAnalytical Chemistry (Washington, DC, United States) (2020), 92 (18), 12400-12406CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society) Horseradish peroxidase (HRP)-based assays feature particular interests because of the simple colorimetric readout. In these assays, 3,3',5,5'-tetramethylbenzidine (TMB) is the most widely used chromogenic substrates for HRP. The later research in nanozyme and DNAzyme also used TMB (the chosen substrate) because they are both HRP-mimics. It should be noted that the substrate of HRP is not just limited to TMB but, in fact, a broad range of benzidine derivs. However, except decreased carcinogenicity due to tetrasubstitution of benzidine, the rationale for the chosen substrate TMB is not clear yet. Here, we addressed such a fundamental issue from the chem. point of view. Nine benzidine derivs. featuring varied properties (different substitution groups and varied no. of substitutions) were selected and investigated with four typical TMB-involved chromogenic systems. Among the existing benzidine substrates that are used for peroxidase-based assays, TMB exhibited the highest sensitivity, better color purity of colored products, and reasonable stability of oxidn. products. Moreover, two tetrasubstituted benzidine derivs. other than TMB (4OCH3 and 2OCH32CH3) were synthesized for comparison. It turned out that the performances (sensitivity, color purity, and stability of the colored products) of TMB are still superior, thus chem. confirming its status of "the chosen substrate" in colorimetric assays. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsF2nsLjO&md5=ad835f23fdd4cfc29da6bec8f4e71b315Lai, W.; Wei, Q.; Zhuang, J.; Lu, M.; Tang, D. Fenton reaction-based colorimetric immunoassay for sensitive detection of brevetoxin B. Biosens. Bioelectron. 2016, 80, 249– 256, DOI: 10.1016/j.bios.2016.01.088 5Fenton reaction-based colorimetric immunoassay for sensitive detection of brevetoxin BLai, Wenqiang; Wei, Qiaohua; Zhuang, Junyang; Lu, Minghua; Tang, DianpingBiosensors & Bioelectronics (2016), 80 (), 249-256CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.) We designed a new colorimetric immunoassay for sensitive monitoring of brevetoxin B (BTB) using enzyme-controlled Fenton reaction with a high-resoln. 3,3',5,5'-tetramethylbenzidine (TMB)-based visual colored system. Upon addn. of hydrogen peroxide (H2O2), the equiv. iron(II) could be first converted into iron(III) and free hydroxyl radical (•OH) via the classical Fenton reaction. Then the as-produced iron(III) and •OH could cause a perceptible change from colorless to blue with the increasing H2O2 concn. in the presence of TMB. Based on Fenton reaction-triggered visual colored system, a novel competitive-type colorimetric enzyme immunoassay was developed for the quant. screening of target BTB on the bovine serum albumin-BTB-modified magnetic bead using glucose oxidase/anti-BTB antibody-labeled gold nanoparticle as the signal-transduction tag. Upon target BTB introduction, the analyte competed with the conjugated BTB on the magnetic bead for anti-BTB antibody on gold nanoparticle. The carried glucose oxidase with the gold nanoparticle could implement the oxidn. of glucose to produce H2O2, and the generated H2O2 promoted the above-mentioned Fenton reaction for color development. Under the optimal conditions, the absorbance decreased with the increasing target BTB in the range from 0.1 to 150 ng kg-1 with a low detection limit (LOD) of 0.076 ng kg-1. The LOD was 500-fold lower than that of commercialized Abraxis BTB ELISA kit. Non-specific adsorption was not obsd. The precision, reproducibility and specificity were acceptable. Finally, the method accuracy was also validated for monitoring spiked seafood samples, giving results well matched with the referenced brevetoxin ELISA kit. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xit1SgtLg%253D&md5=eed2c339abf42a041d37ef48c38357746Robert, A.; Meunier, B. How to Define a Nanozyme. ACS Nano 2022, 16 (5), 6956– 6959, DOI: 10.1021/acsnano.2c02966 6How to Define a NanozymeRobert, Anne; Meunier, BernardACS Nano (2022), 16 (5), 6956-6959CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society) A review. Over the past 15 years, many articles have considered "nanozymes" as ferromagnetic nanoparticles having an "intrinsic peroxidase-like activity" in the presence of hydrogen peroxide. However, the definition and the catalytic activity of these nanozymes have been questioned. The present Perspective reports the main criteria that are essential to classify a nanoparticle as a nanozyme. It is important to consider that not all nanoparticles able to generate hydroxyl radicals in the presence of hydrogen peroxide without catalytic activity can be registered as nanozymes. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xht1Sgs7vL&md5=457641db0f966c8feb07109766629b387Wei, H.; Gao, L.; Fan, K.; Liu, J.; He, J.; Qu, X.; Dong, S.; Wang, E.; Yan, X. Nanozymes: A clear definition with fuzzy edges. Nano Today 2021, 40, 101269, DOI: 10.1016/j.nantod.2021.101269 7Nanozymes: A clear definition with fuzzy edgesWei, Hui; Gao, Lizeng; Fan, Kelong; Liu, Juewen; He, Jiuyang; Qu, Xiaogang; Dong, Shaojun; Wang, Erkang; Yan, XiyunNano Today (2021), 40 (), 101269CODEN: NTAOCG; ISSN:1748-0132. (Elsevier Ltd.) Most nanozyme research has been driven by applications, where the simple goal is the replacement of natural enzymes with more stable, cost-effective and sometimes more active nanomaterials. While in such work, nanozymes can certainly be called catalytic nanomaterials, we believe the conceptualization of these research efforts was encouraged by the name of nanozyme. nanozymes with HRP-like activity have been widely used in ELISA. The nanozyme-based ELISA had a detection limit of 0.67 pg/mL, which is approx. 110-fold better than the HRP-based ELISA. Natural enzymes often suffer from a lack of stability, preventing applications in harsh environments such as sea water or gastric acid. Nanozymes on the other hand have shown excellent stability under such conditions. Vanadium pentoxide (V2O5) nanozymes can substitute vanadium haloperoxidases for antifouling applications. Benefiting from the high stability of V2O5 nanowires, the nanozymes were painted on a stainless steel plate fixed to a boat hull and demonstrated effective suppression of biofouling in seawater for up to 60 days. Studies have also shown that nanozymes fulfill the physiol. function of natural enzymes both on the cellular and animal levels. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvVKgurrK&md5=c9f1c526bf4e2751cd12d3ec22b57f538Zhang, L.; Du, J. Selective sensing of submicromolar iron(III) with 3,3′,5,5′-tetramethylbenzidine as a chromogenic probe. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 2016, 158, 24– 28, DOI: 10.1016/j.saa.2016.01.012 There is no corresponding record for this reference.(a) Chen, L.; Li, Y.; Sun, P.; Chen, H.; Li, H.; Liu, J.; Chen, Z.; Wang, B. A Facile Colorimetric Method for Ultra-rapid and Sensitive Detection of Copper Ions in Water. Journal of Inorganic and Organometallic Polymers and Materials 2022, 32 (7), 2473– 2481, DOI: 10.1007/s10904-022-02255-7 There is no corresponding record for this reference.9Koppenol, W. H.; Liebman, J. F. The oxidizing nature of the hydroxyl radical. A comparison with the ferryl ion (FeO2+). J. Phys. Chem. 1984, 88 (1), 99– 101, DOI: 10.1021/j150645a024 9The oxidizing nature of the hydroxyl radical. A comparison with the ferryl ion (FeO2+)Koppenol, W. H.; Liebman, Joel F.Journal of Physical Chemistry (1984), 88 (1), 99-101CODEN: JPCHAX; ISSN:0022-3654. For the std. redn. potential of the hydroxyl radical/hydroxide couple, 2 values are found in the literature, 2.0 and 1.4 V. Thermochem. data yield 1.98 V for E°(·OHg/OH-aq). Hydration of the hydroxyl radical by -5 kcal/mol changes this value to 1.77 V for E°'(·OHaq/OH-aq), which is still much higher than 1.4 V. It is concluded that the latter value is incorrect. The following thermodn. quantities are derived from, or consistent with, the new E°' value: ΔG°f(·OHaq) = +3.2 kcal/mol; E°'(·OHaq/H2OI) = 2.59 V at pH 0; ΔG°f(O-·aq) = 19.5 kcal/mol; E°'(O-·aq/OH-aq) = 1.64 V at pH 14; ΔG°f(O3-·aq) = 11.7 kcal/mol; E°(O3g/O3-·aq) = 1.19 V; and E°'(H2O2aq/·OHaq, H2OI) = 0.46 V at pH 7. The redn. of hydrogen peroxide by ferrous complexes might yield the ferryl ion (FeO2+-chelate). If the chelating agent is a porphyrin (compd. II) the redn. potential of the couple ferryl/ferriporphyrin is estd. to be 0.9 V. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXktFGlsw%253D%253D&md5=d64f4af2d14cc6cd20b0dee78f74028e10Bard, A. J.; Parsons, R.; Jordan, J. Standard Potentials in Aqueous Solution; Taylor & Francis, 1985. DOI: 10.1201/9780203738764 .There is no corresponding record for this reference.