Substrate For Horseradish Peroxidase Research Articles

Spatiotemporal Proximity-Enhanced Biocatalytic Cascades Within Metal-Organic Frameworks for Wearable and Theranostic Applications.

Enzymatic catalysis, particularly multi-enzyme cascade catalytic, is often limited by the spatial and temporal separation of enzymes and their signal substrates. Herein, a facile method for producing a spatiotemporal proximity-enhanced biocatalytic cascade system is introduced by encasing enzymes within metal-organic frameworks (MOFs) that are modulated with sulfonic acid-functionalized signal substrates. The modulated behavior relies on the sulfonic acid groups coordinated with Zn2+. As a proof of concept, by utilizing 2,2'-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS), a widely-used signal substrate for horseradish peroxidase, two-enzyme/substrate, and three-enzyme/substrate MOFs, which demonstrated a 7.4- and 10.2-fold increase in biocatalytic efficiency over free systems are successfully synthesized. Incorporating the synthesized MOFs into homemade wearable patches and in vivo settings, noninvasive sweat glucose colorimetric detection and photoacoustic imaging-guided photothermal tumor therapy are enabled, respectively. This advancement stems from the newly established coordinative bonds between Zn2+ centers and substrates' sulfonic acid groups, which negates the need for additional signal substrates, thereby not only enhancing but also streamlining bioapplication processes.

Registration of activity of a single molecule of horseradish peroxidase using a detector based on a solid-state nanopore.

This work demonstrates the use of a solid-state nanopore detector to monitor the activity of a single molecule of a model enzyme, horseradish peroxidase (HRP). This detector includes a measuring cell, which is divided into cis- and trans- chambers by a silicon nitride chip (SiN structure) with a nanopore of 5 nm in diameter. To entrap a single HRP molecule into the nanopore, an electrode had been placed into the cis-chamber; HRP solution was added into this chamber after application of a negative voltage. The reaction of the HRP substrate, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), oxidation by the enzyme molecule was performed in the presence of hydrogen peroxide. During this reaction, the functioning of a single HRP molecule, entrapped in the nanopore, was monitored by recording the time dependence of the ion current flowing through the nanopore. The approach proposed in our work is applicable for further studies of functioning of various enzymes at the level of single molecules, and this is an important step in the development of single-molecule enzymology.

Signal Amplification and Near-Infrared Translation of Enzymatic Reactions by Nanosensors.

Enzymatic reactions are used to detect analytes in a range of biochemical methods. To measure the presence of an analyte, the enzyme is conjugated to a recognition unit and converts a substrate into a (colored) product that is detectable by visible (VIS) light. Thus, the lowest enzymatic turnover that can be detected sets a limit on sensitivity. Here, we report that substrates and products of horseradish peroxidase (HRP) and β-galactosidase change the near-infrared (NIR) fluorescence of (bio)polymer modified single-walled carbon nanotubes (SWCNTs). They translate a VIS signal into a beneficial NIR signal. Moreover, the affinity of the nanosensors leads to a higher effective local concentration of the reactants. This causes a non-linear sensor-based signal amplification and translation (SENSAT). We find signal enhancement up to ≈120x for the HRP substrate p-phenylenediamine (PPD), which means that reactions below the limit of detection in the VIS can be followed in the NIR (≈1000 nm). The approach is also applicable to other substrates such as 3,3'-5,5'-tetramethylbenzidine (TMB). An adsorption-based theoretical model fits the observed signals and corroborates the sensor-based enhancement mechanism. This approach can be used to amplify signals, translate them into the NIR and increase sensitivity of biochemical assays.

Signalverstärkung und Nah‐Infrarot Übersetzung von Enzymatischen Reaktionen mit Nanosensoren**

AbstractEnzymatische Reaktionen kommen in zahlreichen biochemischen Verfahren zum Nachweis von Analyten zum Einsatz. Um das Vorhandensein eines Analyten zu messen, werden die Enzyme an eine Erkennungseinheit konjugiert und wandeln ein Substrat in ein (farbiges) Produkt um, das im sichtbaren Bereich (VIS) des Spektrums nachgewiesen werden kann. Der niedrigste nachweisbare enzymatische Umsatz stellt somit eine Grenze für die Empfindlichkeit dar. Hier zeigen wir, dass Substrate und Produkte der Meerrettichperoxidase (HRP) und der β‐Galaktosidase die Fluoreszenz im nahen Infrarot (NIR) von einwandigen Kohlenstoffnanoröhren (SWCNTs) verändern, wenn diese mit (Bio−)Polymeren modifiziert sind. Durch die Fluoreszenz im NIR übersetzen die SWCNTs ein VIS‐Signal in ein NIR‐Signal. Zudem führt die Affinität der Nanosensoren zu einer höheren effektiven lokalen Konzentration der Reaktanten. Dies bewirkt eine nichtlineare sensorbasierte Signalverstärkung und ‐übersetzung (SENSAT). Für das HRP‐Substrat p‐Phenylendiamin (PPD) ergibt sich eine bis zu ≈120‐fache Signalverstärkung, die es erlaubt, Reaktionen unterhalb der VIS Nachweisgrenze im NIR (≈1000 nm) zu verfolgen. Der Ansatz ist auch auf andere Substrate wie 3,3‐5,5′‐Tetramethylbenzidin (TMB) anwendbar. Ein Adsorptionsmodell erklärt die beobachteten Signaländerung und bestätigt den Verstärkungsmechanismus. Mit Hilfe dieses Ansatzes lassen sich Signale von chromogenen enzymatischen Reaktionsprodukten verstärken, in den NIR‐Bereich übertragen und die Empfindlichkeit biochemischer Assays erhöhen.

A facile fluorescence microplate immunoassay based on an in situ fluorogenic reaction for the detection of two highly toxic anticoagulant rodenticides in food and biological matrix

Bromadiolone and brodifacoum, the most frequently used anticoagulant rodenticides, are highly toxic and pose a threat to public health by causing food poisoning incidents. Here, we developed a fluorescence microplate immunoassay for facile and sensitive detection of bromadiolone and brodifacoum by introducing three commercial chemicals (p-phenylenediamine, polyethyleneimine, H2O2) as a new substrate of horseradish peroxidase and then generating fluorescence signals based on an in situ fluorogenic reaction (detection time within 75 min). This assay exhibited higher efficiency in generating fluorescence signals, thereby exhibiting a 6-fold improvement in sensitivity compared with colorimetric ELISA. The limit of detection was 0.23–0.28 ng/mL (ng/g) for bromadiolone and 0.34–0.61 ng/mL (ng/g) for brodifacoum in corn and human serum, with recovery ratios higher than 82.3 %. These satisfactory results illustrated our proposed assay was a potential tool for food analysis and poisoning diagnosis caused by bromadiolone and brodifacoum.

Developing a fluorescence substrate for HRP-based diagnostic assays with superiorities over the commercial ADHP

The combination of horseradish peroxidase (HRP) and a fluorescence substrate has been attracting great interests in developing sensitive biochemical analysis and immunoassays. 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP or Amplex red) is the most sensitive fluorogenic substrate known for HRP in current market, however, it suffers from some drawbacks, such as non-specific reactivity to carboxylesterase and limited fluorescence stability. In the present study, a novel HRP substrate 10-cyclopropylcarbonyl-dichloro-dihydroxyphenoxazine (AR-2), has been prepared, which exhibited improved sensitivity than ADHP in sensing HRP. Moreover, the fluorescence of AR-2/HRP demonstrated improved tolerance to physiological relevant pH fluctuation as compared to ADHP/HRP. Successful detection of uric acid/urate oxidase reaction indicated excellent application prospect of AR-2/HRP for monitoring H2O2-generating biochemical reactions. More interestingly, an enzyme-linked immunosorbent assay (ELISA) using AR-2 as the fluorescence reporter has been successfully used in detecting IgG against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from human serum samples. Overall, AR-2 exhibits improved performances over the commercial ADHP, which will be an ideal alternative to ADHP in HRP-based fluorescence biochemical analysis and immunoassays.

Development of Chemiluminescent ELISA for Detection of Diisobutyl Phthalate in Water, Lettuce and Aquatic Organisms

The use of plasticizers to improve the quality of plastics widely used for household purposes inevitably leads to an increase in their pollution of food and environmental objects. Diisobutyl phthalate (DiBP) is one of the ortho-substituted phthalic acid esters that negatively affect human health and ecosystems. This work is directed to the development of a chemiluminescent enzyme immunoassay (CL-ELISA) for the determination of diisobutyl phthalate in water and food. Luminol, which is oxidized with hydrogen peroxide in the presence of p-iodophenol as an enhancer, was chosen as the substrate for horseradish peroxidase used as a label in the analysis. For this development, rabbit anti-DiBP polyclonal antibodies were generated and tested with the synthesized hapten–protein conjugate. The developed chemiluminescent ELISA has a detection limit of 1.8 ng/mL; the operating range was 5.0–170.8 ng/mL at a content of 10% methanol in the assay medium. The assay was successfully applied to detect diisobutyl phthalate in lettuce leaves, seafood, and water. When using extraction with methanol and hexane, the recovery of DiBP in samples varies in the range of 76.9–134.2%; for assays in natural waters, the recovery rates are from 79.5 to 113.4%.

Discovering a Dihydrofluorescein Analogue as a Promising Fluorescence Substrate to HRP

Horseradish peroxidase (HRP) combined with its fluorescence substrates is attracting increasing attention for biochemical analysis. Amplex red is the most widely used fluorescence substrate to HRP; however, it suffers from some drawbacks, such as nonspecific responsiveness toward carboxylesterases. Discovering a new small molecular fluorescence substrate with improved sensitivity and selectivity for HRP is thus desired. Herein, three dihydrofluorescein derivatives (DCFHs) are presented to serve as HRP substrates through fluorescence turn-on methods. The most promising one, 2,7-dichloro-9-(2-(hydroxymethyl)phenyl)-9H-xanthene-3,6-diol (DCFH-1), exhibited excellent sensitivity in the detection of HRP. Moreover, DCFH-1 does not respond to carboxylesterase, thus holding advantages over Amplex red. In the further study, the detection reagent in the commercial ELISA kits was replaced with DCFH-1 to establish a new fluorescence ELISA, which works very well in the quantification of inflammatory cytokine biomarkers from in vitro models.

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.

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.

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.

Development of enzyme/titanate nanosheet complex coated with molecularly imprinted polydopamine for colorimetric quercetin assay.

A novel hybrid material, which is an enzyme/inorganic nanosheet complex coated by molecularly imprinted polymer (MIP), was developed, and applied to colorimetric quercetin assay. First, an enzyme/inorganic nanosheet complex was prepared from horseradish peroxidase (HRP) enzyme and titanate nanosheet (TiOx), using electrostatic interactions between them in acetate buffer. In the next place, dopamine self-polymerization was performed in the presence of HRP/TiOx complex with quercetin as a template, to prepare MIP membrane onto the HRP/TiOx complex. After washing process, a new hybrid material, MIP-coated HRP/TiOx complex (MIP-HT) was obtained. MIP-HT adsorbed quercetin efficiently, compared with NIP-HT that is an HRP/TiOx complex coated with non-imprinted polydopamine. MIP-HT showed enzymatic activity for an oxidation reaction of guaiacol, which is a chromogenic substrate of HRP, whereas the enzymatic activity of NIP-HT was significantly suppressed. The amount of brown product, formed by the color reaction, reduced owing to the presence of quercetin in sample solution, and a good liner relationship was observed between the concentration of quercetin and the increment of absorbance at 470nm. The investigation using several biomolecules indicates that MIP-HT has the ability to detect quercetin and its analogues with selectivity. Therefore, MIP-HT shows great promise as a new and attractive material for use in colorimetric assay of quercetin or quercetin analogues.

Hybridoma Screening by Antibody Capture: A High-Throughput Western Blot Assay.

Antibody capture assays are often the easiest and most convenient of the hybridoma screening methods. In this procedure, proteins in solution or in a cell lysate are separated according to size by gel electrophoresis and then transferred by blotting to a nitrocellulose sheet. Antigen bound to the solid substrate is incubated with the primary antibody, and the resultant antibody-antigen complexes are detected by a horseradish peroxidase (HRP)-conjugated secondary antibody and a chemiluminescent substrate for HRP.

Fe(III)-mediated reversible catalytic activity of MoS2 nanozymes for bisphosphonate drug sensing

Peroxidase-like activity of MoS2 quantum dots (QDs) can be reversibly regulated by means of Fe3+/alendronate sodium (ALDS)-induced aggregation/disaggregation of the QDs in solution. Specifically, Fe3+ can selectively aggregate the MoS2 QDs and thus greatly enhance their peroxidase-like activity, while such enhancement can be inhibited in the presence of ALDS owing to the competitive coordination of ALDS with Fe3+. By regulating the enzyme-like activity of MoS2 QDs, different colorimetric signal of a typical substrate of horseradish peroxidase, 3,3΄,5,5΄-tetramethylbenzidine, can be measured in the presence of H2O2. Based on this mechanism, we develop a colorimetric approach for the determination of ALDS and further applied in quality control of pharmaceutical products, utilizing either smartphone or UV–vis spectrometer as a readout. This detection method is rapid and selective, where derivatization of ALDS before detection is not needed. Such a smartphone-based colorimetric detection platform is promising to be applied in point-of-care testing at home, small clinics, or underdeveloped regions.

Preparation of species-specific monoclonal antibody and development of fluorescence immunoassay based on fluorescence resonance energy transfer of carbon dots for accurate and sensitive detection of Alicyclobacillus acidoterrestris in apple juice

The growth and metabolism of Alicyclobacillus acidoterrestris can lead to the spoilage of commercial fruit juice. Existing methods have some drawbacks such as complex sample pretreatment, skilled technician requirement, reduced sensitivity and specificity. Herein, a novel fluorescence immunoassay was developed using a monoclonal antibody (mAb) against A. acidoterrestris as the sensing element and carbon dots (CDs) as the signal response unit. The CDs can be quenched via fluorescence resonance energy transfer (FRET) by the oxidization product of p-phenylenediamine (PPD), a chromogenic substrate of horseradish peroxidase (HRP). This approach showed enhanced accuracy and sensitivity with relatively low limit of detection (LOD) of 6.16 × 102 CFU mL−1. Moreover, apple juice contaminated with 1 CFU mL−1 of A. acidoterrestris can be identified after 24 h enrichment. This fluorescence immunoassay could serve as a powerful tool for laboratory identification and on-site inspection of A. acidoterrestris, reducing the adverse effect on the quality of fruit juice.

Sensitive ratiometric fluorescence assay for detecting xanthine in serum based on the inner filter effect of enzyme-catalyzed oxidation products to silicon nanoparticles.

A new type of fluorescent silicon nanoparticles (SiNPs) were prepared via a facile one-pot hydrothermal method by using N-[3-(trimethoxysilyl)propyl]-ethylenediamine (DAMO) and glucose as reagents, and were subsequently applied to construct a ratiometric fluorescence assay for sensitive and rapid determination of xanthine in human serum. Two catalytic oxidation reactions were employed to induce a fluorescence response of the testing system towards xanthine. Under the catalysis of xanthine oxidase (XOD), xanthine in serum samples was oxidized and produced hydrogen peroxide (H2O2). By utilizingo-phenylenediamine (OPD) as the substrate for horseradish peroxidase (HRP) in the presence of H2O2, fluorescent 2,3-diaminophenazine (DAP) was finally generated. A ratiometric fluorescence assay for xanthine was established by determining the ratio of the green-yellow fluorescence emission of DAP and the blue fluorescence emitted from SiNPs under the inner filter effect (IFE) of DAP. Instead of traditional multi-step procedures for adding reacting reagents to the testing solution, all the reaction reagents were mixed with serum samples in a single step for this assay to shorten the total reaction time. This assay demonstrates superiority over a solo DAP fluorescence-based assay as well as other reported methods, with excellent sensitivity and reduced testing time. The strategies proposed in this work for both synthesis and application of fluorescent SiNPs can be used in future fabrication of novel fluorescent probes, especially for sensing biological metabolites involved in H2O2-generation or consumption reactions.

Synthesis and application of new 3-amino-2-pyridone based luminescent dyes for ELISA

A series of new 3-amino-4-arylpyridin-2(1H)-one derivatives was synthesized on the basis of oxazolo[5,4-b]pyridin-2(1H)-ones. Their photophysical properties and antioxidant activity were studied. Effective luminophores with an photoluminescence yield of up to 0.76 and substances exhibiting antioxidant activity exceeding ascorbic acid were revealed. It was found that the obtained luminescent dyes are horseradish peroxidase substrates and can be used to develop a rather sensitive ELISA method. The detection limits of hydrogen peroxide and horseradish peroxidase in the system containing the tested dyes lie in the nanomolar concentration range.

Rationale of 3,3',5,5'-Tetramethylbenzidine as the Chromogenic Substrate in Colorimetric Analysis.

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 derivatives. 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 chemistry point of view. Nine benzidine derivatives featuring varied properties (different substitution groups and varied number 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 oxidation products. Moreover, two tetrasubstituted benzidine derivatives 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 chemically confirming its status of "the chosen substrate" in colorimetric assays.

DNA methylation modulates allograft survival and acute rejection after renal transplantation by regulating the mTOR pathway

Acute rejection (AR) can lead to allograft dysfunction following renal transplantation, despite immunosuppressive treatments. Accumulating evidence points out a role for epigenetic modification in immune responses. However, the mechanism and contribution of DNA methylation in allograft survival remain unclear. In this study, we followed up patients who successively experienced end‐stage renal disease, renal transplantation with allograft function or dysfunction, and hemodialysis. Peripheral blood mononuclear cells were collected at different time points for analysis of the DNA methylation. Epigenetic modifier analysis was also performed to explore its effect of methylation in a mouse model of AR. Compared with the allograft‐stable cohort, patients who experienced AR‐induced allograft dysfunction demonstrated more changes in methylation patterns. Pathway analysis revealed that the hypermethylated areas in the allograft dysfunction group were associated with genes related to the mechanistic target of rapamycin (mTOR) signaling pathway. Moreover, in the mouse AR model, treatment with the DNA methyltransferase inhibitor—decitabine regulated the Th1/2/17/regulatory T cell (Treg cell) immune response via its demethylating role in the suppressing the activity of the mTOR pathway, which ultimately ameliorated renal allograft‐related inflammatory injuries. These results revealed that changes in methylation accompany AR‐induced allograft dysfunction after renal transplantation. Epigenetics may provide new insights into predicting and improving allograft survival.

Horseradish peroxidase interacts with the cell wall peptidoglycans on oral bacteria.

Salivary peroxidase and myeloperoxidase are known to display antibacterial activity against oral microbes, and previous indications have pointed to the possibility that horseradish peroxidase (HRP) adsorbs onto the membrane of the major oral streptococci, Streptococcus mutans and Streptococcus sanguinis (S. sanguinis). However, the mechanism of interaction between HRP and the bacterial cell wall component is unclear. Dental plaques containing salivary glycoproteins and extracellular microbial products are visualized with 'dental plaque disclosing agent', and are controlled within dental therapy. However, current 'dental plaque disclosing agents' are difficult to evaluate with just dental plaques, since they stain and disclose not only dental plaques but also pellicle formed with salivary glycoproteins on a tooth surface. In this present study, we have demonstrated that HRP interacted with the cell wall component of the major gram-positive bacterial peptidoglycan, but not the major cell wall component of gram-negative bacteria lipopolysaccharide. Furthermore, we observed that the adsorbed HRP labeled with fluorescence was detected on the major oral gram-positive strains S. sanguinis and Streptococcus salivarius (S. salivarius), but not on a gram-negative strain, Escherichia coli (E. coli). Furthermore, we have demonstrated that the combination of HRP and chromogenic substrate clearly disclosed the dental plaques and the biofilm developed by S. sanguinis, S. salivarius and the major gram-postive bacteria Lactobacillus casei on tooth surfaces, and slightly disclosed the biofilm by E. coli. The combination of HRP and chromogenic substrate did not stain either the dental pellicle with the salivary glycoprotein mucin, or naked tooth surfaces. These results have suggested the possibility that the adsorption activity of HRP not only contributes to the evaluation of dental plaque, but that enzymatic activity of HRP may also contribute to improve dental hygiene.