Peroxidase Substrate Research Articles

Dual enzyme-like properties of silver nanoparticles decorated Ag2WO4 nanorods and its application for H2O2 and glucose sensing

The facile one-pot hydrothermal synthesis of silver nanoparticles decorated silver tungstate nanorods (Ag@Ag2WO4 NRs) and their catalytic activities similar to those of natural enzymes catalase and peroxidase were reported. The Ag@Ag2WO4 NRs could catalyze the decomposition reaction of H2O2 into water and oxygen besides catalyzing the reduction of H2O2 into water in the presence of peroxidase substrates. Spectrophotometric and electrochemical methods were used to investigate the pH-dependent dual enzyme mimics exhibited by Ag@Ag2WO4 NRs. The Ag@Ag2WO4 NRs showed a lower Km value when compared to the natural horseradish peroxidase enzyme showing the stronger affinity for hydrogen peroxide and TMB. The peroxidase-like property of the synthesized Ag@Ag2WO4 NRs was exploited to develop a H2O2 sensor with a broad linear range and low detection limit. Thus, a wide linear range of 45.4 μM- 2.38 mM and a low detection limit of 5.4 μM was obtained by spectrophotometry while a wide linear range of 62.34 μM- 2.4 mM and a low detection limit of 6.25 μM was obtained by amperometry for H2O2. Further, the detection method was extended for the detection of glucose with a wide linear range of 27.7 μM- 0.33 mM and a low detection limit of 2.6 μM.

A sensitive colorimetric immunoassay based on poly(dopamine) modified magnetic nanoparticles for meat authentication

Magnetic nanoparticles as solid substrate and polydopamine as coating polymer were combined for the construction of a sensitive colorimetric immunoassay aimed at the quantification of pork associated with meat fraud. In this study, a competitive assay was conducted between purified IgGs and porcine IgGs covalently immobilized on magnetic nanoparticles, for the binding sites of peroxidase-labelled antibody. The recognition of porcine IgG was monitored by a simple colorimetric method in the presence of peroxidase substrate. The performed immunoassay was successfully employed for the screening of pork in adulterated meat, allowing low level of 0.01% to be detected in short time of analysis. The specificity of the immunoassay was tested against five different meat species (pork, lamb, turkey, chicken and beef) and it was found to be highly specific towards pork. Furthermore, the used magnetic probe has maintained a satisfactory stability enabling sensitive detection of pork within 20 days of storage. With the versatility and facile-preparation of polydopamine modified magnetic nanoparticles as well as the easy handling of the whole assay, this colorimetric method provides a specific and sensitive in-field-test for the detection of pork in adulterated meat and thus verifying halal meat authenticity.

A sensitive and reversible staining of proteins on blot membranes

A modified, sensitive and reversible method for protein staining on nitrocellulose (NC) and polyvinylidine fluoride (PVDF) membranes was developed in Western blotting. The method employed Congo red staining to visualize proteins on different blot membranes. Staining of proteins with Congo red dye is more faster procedures. According to the experimental results, approximate 20 ng proteins could be detected in 3 min in room temperature. The staining on the proteins is easily reversible with Congo red destaining solution for NC and PVDF membranes, so that the blot membranes can be reused for Western blotting. In addition, we confirmed that the staining method is fully compatible with Western blot detection. NC and PVDF membranes treatment with Congo red staining does not interfere with conventional chemiluminescent substrates of peroxidase. As compared to MemCode reversible protein stain kits from Pirece Biotechnology, the staining technique is more sensitive, lower of cost, convenient and not adversely affecting subsequent Western blotting results. On the other hand, the stain is more sensitive than the Ponceau S staining. Therefore, Congo red staining is a promising and ideal alternative for current protein stain. Besides, the binding modes of Congo red or Ponceau S stain were investigated using various 2D and 3D molecular docking and demonstrated potential molecular basis for sensitivity of Congo red staining are higher than Ponceau S.

A dual-signal colorimetric and ratiometric fluorescent nanoprobe for enzymatic determination of uric acid by using silicon nanoparticles.

The authors describe a dual-signal colorimetric and ratiometric fluorescent probe for uric acid (UA). It is based on cascade catalysis and an inner filter effect. The method involves uricase-catalyzed oxidation of UA and iodide-catalyzed oxidation of the colorless peroxidase substrate o-phenylenediamine (OPD) to form yellow 2,3-diaminophenazine (oxOPD). This can be visually observed or monitored by measuring absorbance at 417nm. Furthermore, oxOPD quenches the fluorescence of silicon nanoparticles (SiNPs) (with peaks at 450 and 565nm) via an inner filter effect. The change in the ratio of emissions peaking 565 and 450 (at excitation wavelength of 380 nm) increases linearly in the 0.01-0.8mM UA concentration range). The lower detection limits are 8.4 and 0.75μM when using the colorimetric and ratiometric fluorometric method, respectively. The assay was successfully applied to the quantitation of UA in spiked serum samples. Graphical abstractA dual-signal colorimetric and ratiometric fluor ometric assay was developed for uric acid (UA). The fluorometric assay is based on the inner filter effect between fluorescent silicon nanoparticles and 2,3-diaminophenazine. It involves uricase-catalyzed oxidation of UA, and iodide-catalyzed oxidation of o-phenylenediamine.

A compact nanobody-DNAzyme conjugate enables antigen detection and signal amplification

Hemin-utilizing G-quadruplex DNAzymes with peroxidase-like (POX) activity are widely used as signal reporters in biosensing technology. However, their application to protein detection has been mostly limited to sandwich-type assays involving streptavidin or nanoparticles as indirect bridging platforms between DNAzymes and antibodies. Herein, we describe the generation of a compact, covalently DNAzyme-labeled nanobody which was successfully tested in a direct enzyme-linked immunosorbent assay (ELISA). The conjugation approach was based on the self-labeling protein tag mVirD2, a truncated bacterial relaxase able to covalently bind DNA with 1:1 stoichiometry at a specific amino acid residue. The hybrid molecule combined the nanobody antigen binding affinity and specificity with the DNAzyme catalytic capability to oxidize peroxidase substrates (e.g. ABTS, H2O2). The proposed strategy is simple and cost-effective, enables development into multiplex formats and provides reagents with hitherto unmet reproducibility in terms of POX activity instrumental for both colorimetric and electrochemical reactions. As a proof-of-concept, it was demonstrated that DNAzyme-nanobody conjugates are convenient immunoreagents for rapid and specific detection of the toxic alga Alexandrium minutum.

Dual responsive magnetic Fe3O4-TiO2/graphene nanocomposite as an artificial nanozyme for the colorimetric detection and photodegradation of pesticide in an aqueous medium

The Fe3O4-TiO2/reduced graphene oxide (Fe3O4-TiO2/rGO) nanocomposite was successfully prepared by one step hydrothermal method and exhibit intrinsic peroxidase mimic activity and photocatalytic efficiency. The as-prepared nanomaterials were characterized by several analytical tools including XRD, HRTEM, FESEM, XPS, VSM, FT-IR, AFM, TGA and zeta potential analysis. The average particle size of Fe3O4 and TiO2 NPs on the rGO nanosheets are found to be 9 ± 0.2 nm. The synthesized nanocomposite showed dual responsive including highly sensitive colorimetric detection of harmful atrazine pesticide in an aqueous medium as well as photocatalytic degradation of atrazine pesticide. The Fe3O4-TiO2/rGO nanocomposite showed the efficient peroxidase-like catalytic activity throughout the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) as a peroxidase substrate to the blue-colored oxidized product (ox-TMB) in presence of H2O2. Based on this observation, the colorimetric detection technique is applied for the sensing of atrazine as model pesticides using TMB as a peroxidase substrate molecule and 2.98 μg/L of the limit of detection (LOD) was obtained in the linear range of 2–20 μg/L. Thus the proposed colorimetric sensing technique is simple and low cost for the real-time monitoring of the pesticides in an aqueous medium. Further, the Fe3O4-TiO2/rGO nanocomposite was also successfully utilized towards efficient photocatalytic degradation of atrazine molecule (100 %) under irradiation of natural sunlight. Moreover, Fe3O4-TiO2/rGO nanocomposite was successfully recycled for 10 times without a significant loss of its photocatalytic efficiency. This work delivers a new insight for the dual responsive of the Fe3O4-TiO2/rGO nanocomposite as an artificial nanozyme for colorimetric sensing of the water pollutant and also removal of the water pollutant by simple photocatalytic degradation method under natural sunlight irradiation.

ATP Mimics pH-Dependent Dual Peroxidase-Catalase Activities Driving H 2 O 2 Decomposition

Adenosine triphosphate (ATP) is produced mainly in the mitochondrion, and its primary task is to function as a ubiquitous energy currency to meet the cellular metabolic demands in biological system...

Acoustic Histology with Specific Dyes and Antibodies

The present study aims to identify specific staining methods for acoustic histology. We compared attenuation-of-sound (AOS) images from scanning acoustic microscopy (SAM) with light microscopy (LM) images. Ethanol-fixed tissue or cytology samples and formalin-fixed surgical or autopsy specimens were examined. Nuclei, collagen, elastic fibers and polysaccharides and various antigens, including cell surface, cytoplasmic, nuclear and stromal substances, were observed. Samples with various fixation methods were used. Hematoxylin staining had significantly higher AOS values in accordance with staining duration. Specific staining for collagen, elastic fibers and polysaccharides increased the AOS values of the specific substance. Using diaminobenzidine tetrahydrochloride in NiCl2 solution as a substrate for horseradish peroxidase increased the AOS values to those suitable for acoustic immunostaining. Collagenase digestion after collagen staining decreased AOS values, reflecting collagen density and distribution. Staining with specific dyes or acoustic immunostaining enabled the histologic localization of specific substances by SAM, similar to LM.

Using target-specific aptamers to enhance the peroxidase-like activity of gold nanoclusters for colorimetric detection of tetracycline antibiotics

Tetracycline antibiotics (TCs) are one kind of broad spectrum bacteriostatic agents. However, excessive use of TCs will have a threat to the environment and human health. Therefore, it is necessary to develop a simple method for direct detection of TCs. Based on intrinsic peroxidase-like activity of gold nanoclusters (AuNCs), we used TC-specific aptamers (Apt) to improve the catalytic activity of AuNCs toward the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation by H2O2, and established a colorimetric sensing platform for TCs. The catalytic enhancement by Apt allows for sensitive colorimetric detection of TCs, and Apt as molecular recognition elements can specifically combine with TCs leading to high selectivity. This developed sensing platform can quantitatively detect TCs in the concentration range of 1–16 μM with a limit of detection (LOD) as low as 46 nM. Interestingly, the naked-eye detection capability of this method is estimated to be 0.5 μM. Finally, the detection of TCs in real samples like drugs and milk was validated.

Counter Anion-Directed Growth of Iron Oxide Nanorods in a Polyol Medium with Efficient Peroxidase-Mimicking Activity for Degradation of Dyes in Contaminated Water.

Development of nanozymes, which are nanomaterials with intrinsic enzymatic properties, has emerged as an appealing alternative to the natural enzymes with tremendous application potential from the chemical industry to biomedicine. The self-assembled growth of micrometer-sized oxide materials with controlled nonspherical shapes can be an important tool for enhancing activity as artificial enzymes, as the formation of these superstructures often results in high surface area with favorable impact on catalytic activity. Herein, the growth of rod-shaped Fe3O4 microstructures via a one-pot microwave-based method and using a water–poly(ethylene glycol) mixture as a solvent is reported, without the involvement of external shape-directing agents. The precursor metal salt played a key role in the size, shape, and phase selective evolution of iron oxide micro/nanomaterials. Whereas self-assembled microrod superstructures were obtained using Fe(NO3)3 as the metal salt precursor, use of FeCl3 or Fe-acetate as precursors afforded hollow Fe2O3 microparticles and Fe3O4 nanoparticles, respectively. A graphitic layer was deposited on the Fe3O4 surface, imparting a negative surface charge as a result of a high-temperature treatment of poly(ethylene glycol). The rod-shaped Fe3O4 microcrystals show efficient peroxidase-mimicking activity toward 3,3,5,5′-tetramethylbenzidine and pyrogallol as peroxidase substrates with a Michaelis–Menten rate constant (Km) value of 0.05 and 0.52 mM, respectively. The proficient enzyme mimicking behavior of these magnetic superstructures was further explored for the degradation of organic dyes that includes rhodamine B, methylene blue, and methyl orange with a rate constant (k) of 0.038, 0.011, and 0.007 min–1 respectively, using H2O2. This fast and simple method could help to develop a new pathway for differently shaped oxide nanoparticles in a sustainable and economical manner that can be harnessed as nanozymes for industrial as well as biological applications.

Bioinspired hierarchical CoAl-LDH/MFe2O4(Ni, Zn, Co) as peroxidase mimics for colorimetric detection of glucose

Novel hierarchically porous CoAl-LDH (layered double hydroxides)/MFe2O4(Ni, Zn, Co) hybrids with excellent peroxidase-like catalytic activity, high selectivity and reusability were first fabricated by using natural cotton as a bio-template. Among them, CoAl-LDH/NiFe2O4 exhibited the best intrinsic peroxidase-like property due to its 3D hierarchically porous structure with more exposed active sites, which could effectively catalyze peroxidase substrate 3,3′,5,5′- tetramethylbenzidine (TMB) in the presence of H2O2 to produce typical blue color reaction. A facile and visual system for glucose detection using glucose oxidase was successfully established, which exhibited a sensitive response to glucose in the range of 25–400 μM with detection limit of 13 μM based on the hydroxyl radical originated from the decomposition of H2O2 by the catalysis of CoAl-LDH/MFe2O4(Ni, Co, Zn) hybrids. This work offered an effective strategy to achieve morphology-controllable hierarchically porous materials and provided a new insight into seeking novel enzyme mimetics with enhanced catalytic activities.

3D-printed rolling circle amplification chip for on-site colorimetric detection of inorganic mercury in drinking water

A point-of-care testing chip was developed for the colorimetric detection of inorganic mercury ion (Hg2+). The disposable chip fabricated by three-dimensional printing technology contains DNAzymes produced by rolling circle amplification (RCA); a color change caused by the enzymatic reaction between DNAzymes and the peroxidase substrate 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) is measured using a portable spectrophotometer. In the “turn-off”-type RCA reaction, the annealing of the T(12) primer that initiates the RCA reaction is blocked by the interaction of thymine with Hg2+; thus, the amount of amplified DNAzymes causing a color change is decreased depending on Hg2+ concentration. The colorimetric signal is enhanced by amplifying double-repeat DNAzymes from a circular DNA template. The chip detected Hg2+ in tap drinking water samples with high sensitivity (lowest validated value: 3.6 μg/L) and showed better selectivity, precision, and reproducibility than conventional analysis instruments. This low-cost easy-to-use platform can reduce the risk of accidental Hg2+ poisoning.

Preparation of porous uranium oxide hollow nanospheres with peroxidase mimicking activity: application to the colorimetric determination of tin(II).

Porous uranium oxide hollow sphere nanoparticles were synthesized in ionic liquids under hydrothermal conditions. Various precipitating agents and ionic liquids were investigated to determine their respective impact on the resultant uranium oxide morphologies. Using hydrazine hydrate as precipitating agent and N-butyl pyridinium bromide as templating agent, aporous-hollow structure was created with a surface area of 1958 m2.g-1 and an average pore diameter of 30nm. The nanoparticles revealed high peroxidase-mimicking activity. This was evaluated by using the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) that is catalytically oxidized by H2O2 to give oxidized TMB (oxTMB) which is blue (with an absorption peak at 652nm). The material was used as a nanozyme for colorimetric detection of Sn2+. Meanwhile, it is found that BSA strongly improves the catalytic activity of the nanozyme, while Sn(II) inhibits its activity. Thus, a colorimetric method for Sn2+ detection was designed. The method works in the 0.5-100μM Sn(II) concentration range and has a lower detection limit of 0.36μM (at S/N = 3). Graphical abstract The catalytic activity of porous-hollow nano-UO2 toward the oxidation of 3,3',5,5'-tetramethylbenzidine by H2O2 is remarkably improved in the presence of bovine serum albumin, while tin(II) inhibits its activity. This finding has been applied to design a method for colorimetric quantification of tin(II) in water samples.

Bivalent Metal Ions Tethered Fluorescent Gold Nanoparticles as a Reusable Peroxidase Mimic Nanozyme

Nanozymes are a class of mimic enzymes that have both unique properties of nanomaterials and catalytic functions. They are widely used in medicine, food, agriculture, and environment due to their high catalytic efficiency, stability, economy, and large-scale preparation. In this work, a simple, gentle method was applied to synthesize adenosine phosphates (AXP) templated gold nanoparticles (Au NPs) with small size, good optical properties, and peroxidase-like activities. In the presence of hydrogen peroxide (H2O2), the peroxidase substrate 3, 3′, 5, 5′-tetramethylbenzidine (TMB) could be oxidized to form blue-colored oxidized TMB (oxTMB), which demonstrates that the AuAXP NPs have peroxidase-like activities. Interestingly, the introduction of bivalent metal ions into AuAXP NPs allows their reusability. Among them, after three rounds of recycling, the catalytic activity of Mg@AuAMP could still reach 92.69% compared to the original level, which showed excellent reusability. In addition, the glutathione (GSH) detection was realized with a linear detection range of 0–200 μM due to the GSH-induced loss of peroxidase-like activities. Overall, this work provides a new idea of recycling for biosensor and catalytic analysis.

AuCu bimetal nanoclusters as high-performance mimics for ultrasensitive recognition of biomolecules

Introducing novel mimic materials as alternatives for natural enzymes challenges the analysts. Study on the peroxidase-like materials is an active field in analytical research areas. Herein, Au/Cu bimetal nanoclusters (Au/Cu NCs) are introduced as highly efficient peroxidase mimics, which were investigated using fluorometric and colorimetric techniques. A comprehensive comparison between the catalytic activity of Au, Cu, and their bimetal NCs, with different ratios of Au/Cu was performed using some different peroxidase substrates (including 3,3′,5,5′-tetramethylbenzidine (TMB), o-phenylenediamine dihydrochloride (OPD), and terephthalic acid (TA)). Additionally, different capping agents were applied for the synthesis of NCs, and it was found that penicillamine-capped NCs with 50% Cu have higher activity than other synthesized NCs. Analytical application of the novel mimic for H2O2 detection caused a linear calibration in a wide linear range of 0.001–3 μmol/L, and a great detection limit (3S) of 0.18 nmol/L, using a sensitive fluorescence system. The developed system was also sensitive for recognizing glucose and cholesterol in blood samples, after their enzymatic oxidation and production of H2O2. Detection limits of 55 and 15 nmol/L were obtained for glucose and cholesterol, respectively. The presented method also showed good reliability, which was validated by certified reference materials.

Porous Ruthenium Selenide Nanoparticle as a Peroxidase Mimic for Glucose Bioassay

Nanozyme is a promising field that offers the substitution for natural enzymes using various nanomaterials. Various nanomaterials with peroxidase-like activity were investigated. Among them, transition metal chalcogenides were explored as promising nanozymes due to their excellent enzyme-mimicking activities. However, ruthenium selenide has not been studied as a peroxidase mimic because of the difficulty for synthesis. Herein, we prepared ruthenium selenide nanomaterial with ordered mesoporous structure (P-RuSe2) employing KIT-6 silica as the template. The composition and structure of P-RuSe2 were fully characterized. Further, its peroxidase-like activity was investigated. P-RuSe2 possessed excellent peroxidase-mimicking activity, which catalyzed the oxidation of peroxidase substrates, including 3,3′,5,5′-tetramethylbenzidine, o-phenylenediamine, and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) in the presence of H2O2. Moreover, P-RuSe2 exhibited higher peroxidase-like activity when compared with several representative nanozymes as well as bulk RuSe2. To demonstrate its potential applications, the colorimetric detection systems for H2O2 and glucose were successfully constructed based on P-RuSe2 nanozyme.

Transferrin-conjugated quasi-cubic SPIONs for cellular receptor profiling and detection of brain cancer

The high biocompatibility, unique magnetic properties and significant biomedical potential of superparamagnetic iron oxide nanoparticles (SPIONs) have spurred a major interest in developing new strategies to synthesise aqueous-phase dispersible SPIONs. In parallel, the rapidly growing field of enzyme-mimicking catalytic nanomaterials, referred to as NanoZymes, has seen immense interest in utilising the unique properties of NanoZymes for new applications. Here, we report a new strategy for the large-scale synthesis of water dispersible quasi-cubic γ-Fe2O3 SPIONs. This strategy provides a unique way to control the morphological characteristics of SPIONs through the in-situ formation of sodium oleate complex. Notably, our approach eliminates the need for high temperatures, complex cleaning protocols and post-synthesis phase-transfer strategies that are typically required during the well-established thermal decomposition synthesis approaches. We further establish the peroxidase-mimic NanoZyme activity of these quasi-cubic SPIONs and use them as an effective transducer platform to generate a unique colour through efficient oxidation of peroxidase substrates. By combining these inorganic enzymes with transferrin (Tf) as a brain cancer-specific targeting agent, we create a simple yet effective colorimetric biosensing platform that allows us to monitor the cellular expression profile of transferrin receptors (TfR). The difference in the expression profile of TfR in the U87MG glioblastoma cells and the fibroblast cells generates a distinct tonality of the colorimetric response. This not only allows cell surface receptor profiling but also allows robust identification of brain cancer cells. The high accuracy and precision of the proposed biosensor outlines the robustness of the NanoZyme-based catalysts in colorimetric detection of various targets.

Enhancing the peroxidase-like activity of ficin by rational blocking thiol groups for colorimetric detection of biothiols

The peroxidase-like activity of ficin is relatively low, which limits its application. It was found that thiol groups of ficin could inhibit its peroxidase-like activity. So, two procedures, i.e., direct blocking with N-ethylmaleimide (NEM), or using tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to interrupt disulfide bonds then blocking thiol groups with NEM, were applied to block thiol groups of ficin, ficin-NEM (ficin-N) and ficin-TCEP-NEM (ficin-TN) were produced, respectively. The blocking of thiol groups accelerated the peroxidase activity dramatically. The peroxidase catalytic activity of ficin-N and ficin-TN toward the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation by H2O2 was about 2.5-fold and 5-fold increase compared with ficin, respectively, which accompanied a color change from colorless to blue and followed classic Michaelis-Menten model. The kinetic parameters indicated that higher affinity of ficin-N (Km = 0.31) and ficin-TN (Km = 0.39) to H2O2 compared with ficin (Km = 0.58), and ficin-TN had the highest Kcat which increased by 6.5 times and 4.5 times for TMB and H2O2, respectively. According to these findings, a colorimetric method with high sensitivity for the detection of biothiols was developed due to sulfhydryl compounds inhibited the peroxidase activity of ficin. Comparing with ficin and ficin-N, ficin-TN had the widest detection range (0.01–16 μM) and the lowest detection limit (3 nM). The practical applications of ficin-TN for biothiol determination in human serum samples have been demonstrated with satisfactory results. Ficin-N and ficin-TN are promising to apply to the bioanalysis.

Colorimetric sensing for ascorbic acid based on peroxidase-like of GoldMag nanocomposites.

A new scheme for sensitive and rapid colorimetric detection of ascorbic acid (AA) has been developed by the GoldMag-ABTS free radical scavenging system. The well-dispersed Gold and magnetic particles (GoldMag) was successfully prepared by self-assembly method and characterized by Fourier transform infrared (FTIR), X-Ray Photoelectron Spectroscopic (XPS) techniques. Nanocomposites combine the advantages of superparamagnetic, biocompatibility and high catalytic activity of Fe3O4 and gold nanoparticles (AuNPs) and exhibit enhanced the intrinsic peroxidase-like activity, which can be used to catalyze the oxidation of the peroxidase substrates 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) produce a green-colored product in presence of hydrogen peroxide. Ascorbic acid as an effective antioxidant have scavenging effects on ABTS radicals and induce the reduction of green ABTS.+ to colorless ABTS2-, resulting in a significant green color fading. On this basis, a rapid, sensitive and selective colorimetric assay for ascorbic acid has been developed. Under optimal conditions, ascorbic acid has a linear response range from 0.01 mmol/L to 1 mmol/L with a detection limit of 0.12 μmol/L and a short assay time of the 30 s. Furthermore, the colorimetric system showed good sensitivity, stability, selectivity, and repeatability. It also successfully applied to the determination of ascorbic acid in real samples.

OPTIMIZATION OF DIAGNOSTIC IMMUNOHISTOCHEMISTRY OF FORMALIN-fiXED, PARAffiN-EMBEDDED TISSUES

Objective Immunohistochemistry (IHC) is a widely used diagnostic technique in the Oral and Maxillofacial Pathology. Various variables affect results of the IHC technique. Therefore, standardization and optimization of the IHC technique are essential in generating reliable and reproducible results. The aim of this study was to determine the optimal conditions for IHC staining of multiple antibodies with minimal background. Findings In our study, we noticed that 2% hydrogen peroxide (H2O2) is most effective to block endogenous peroxidase activity. Formalin-fixed, Paraffin-embedded tissues from mice and human biopsies were subjected to IHC with three different monoclonal chimeric antibodies. Various combinations of antibody concentrations and incubation time were investigated. Two secondary antibody kits namely Vectastain Universal ABC-AP KIT (PK-6200) and Alkaline Phosphatase Universal (AK-5200) as well as two chromogen systems namely ImmPACT DAB EqV (Chromogen and peroxide) and Alkaline Phosphatase substrates were used. The optimal concentration of individual antibodies varied greatly (From 1 to 20 µg/ml) based on their affinity to the primary antigen. While the manufacturer instructions recommend 30 minutes incubation for all primary antibodies, we observed overnight incubation at 4oC obtained best results. The optimal counterstain with peroxidase (brown) substrate was Hematoxylin and Alkaline Phosphatase (blue) substrate was Nuclear Fast Red. Digital imaging parameters such as white balance, exposure time and file format were optimized. Evaluation of the IHC results was performed using the light microscope and digital imaging. Conclusion Overall, our results confirm careful validation of individual IHC technique is critical in obtaining consistent and reproducible results. Factors such as endogenous peroxidase blocking, antibody concentration and incubation time, chromogen system and counterstains are important parameters that should be optimized for individual studies.