Optimization of heterocyclic 4-hydroxystyryl derivatives for histological localization of endogenous and immunobound peroxidase activity

Assisted by the development of light excitation and measuring techniques and the commercial availability of highly sensitive equipment, luminescent labels are sensitive detection tools for life sciences research. By contrast to a wide variety of well established chromogenic techniques, fluorescent labels for detecting peroxidase (PO) have been confined to only a few substrates. We describe here novel fluorescent substrates of PO derived from heterocyclic 4-hydroxy styrenes as useful tools for detecting endogenous and exogenous targets in fixed cells and tissues. Excellent localization, high staining sensitivity, outstanding photostability, and exceptionally low background staining were achieved by optimizing the substrate through chemical synthesis. Structure/staining behavior relationships are discussed. By contrast to tyramine-fluorochrome conjugates employed in the catalyzed reporter deposition (CARD) technique, reporting and anchoring functions are no longer separated. Consequently, enzymatic cross-linking of the substrate yields an altered fluorochrome with different properties. Spectral properties and anchoring capability are interdependent and influenced by environmental effects and pH. We screened overall staining capability of 4-hydroxy styryl derivatives using an iterative semi-empirical approach, and ascertained optimal substitution patterns for high PO staining specificity and high fluorescence response. Reliable staining performance was achieved with alkyl chains of short or medium length at the positively charged nitrogen, whereas introducing polar groups often impaired the staining specificity of PO. Catalytic cross-linking of heterocyclic 4-hydroxy-styryl derivatives is a promising approach for permanent fluorescent staining of PO in fixed cells and tissues, and complements the CARD technique. Histochemical and immunohistochemical applications are presented using conventional and confocal fluorescence microscopes with different excitation sources. Spectral properties of selected stains are discussed. Novel stains also are of potential interest as “reactive-tracers” for living cells under multi-photon laser excitation conditions, because they exhibit pronounced nonlinear optical properties.     

Optimization of heterocyclic 4-hydroxystyryl derivatives for histological localization of endogenous and immunobound peroxidase activity

Assisted by the development of light excitation and measuring techniques and the commercial availability of highly sensitive equipment, luminescent labels are sensitive detection tools for life sciences research. By contrast to a wide variety of well established chromogenic techniques, fluorescent labels for detecting peroxidase (PO) have been confined to only a few substrates. We describe here novel fluorescent substrates of PO derived from heterocyclic 4-hydroxy styrenes as useful tools for detecting endogenous and exogenous targets in fixed cells and tissues. Excellent localization, high staining sensitivity, outstanding photostability, and exceptionally low background staining were achieved by optimizing the substrate through chemical synthesis. Structure/staining behavior relationships are discussed. By contrast to tyramine-fluorochrome conjugates employed in the catalyzed reporter deposition (CARD) technique, reporting and anchoring functions are no longer separated. Consequently, enzymatic cross-linking of the substrate yields an altered fluorochrome with different properties. Spectral properties and anchoring capability are interdependent and influenced by environmental effects and pH. We screened overall staining capability of 4-hydroxy styryl derivatives using an iterative semi-empirical approach, and ascertained optimal substitution patterns for high PO staining specificity and high fluorescence response. Reliable staining performance was achieved with alkyl chains of short or medium length at the positively charged nitrogen, whereas introducing polar groups often impaired the staining specificity of PO. Catalytic cross-linking of heterocyclic 4-hydroxy-styryl derivatives is a promising approach for permanent fluorescent staining of PO in fixed cells and tissues, and complements the CARD technique. Histochemical and immunohistochemical applications are presented using conventional and confocal fluorescence microscopes with different excitation sources. Spectral properties of selected stains are discussed. Novel stains also are of potential interest as “reactive-tracers” for living cells under multi-photon laser excitation conditions, because they exhibit pronounced nonlinear optical properties.     

<Emphasis Type="Italic">N,N</Emphasis>-Dialkylaminostyryl dyes: specific and highly fluorescent substrates of peroxidase and their application in histochemistry

Fluorescent labeling of immuno-bound or endogenous peroxidase (PO) activity has been achieved to date by means of phenol derivatives with a low substitution degree. Here it is demonstrated that N,N-dialkylamino-styryl dyes can also act as fluorescent substrates of PO. They undergo enzymatically cross-linking reactions to surrounding cell constituents in an analogous manner thus permitting highly fluorescent and permanent labeling. This approach is narrowly related to the catalyzed reporter deposition (CARD) technique based on tyramine conjugates and the recently described catalytic cross-linking approach of hydroxystyryl derivatives. The substitution patterns for optimal cross-linking capability and the spectral properties of obtained specific reaction products were studied using an iterative semi-empirical approach. The best staining performance is achieved with N,N-dimethylaminoaryl derivatives. Their N,N-dialkyl homologues as well as the primary aryl amine pendants failed as PO substrates. Due to their basic character, novel substrates occasionally tend to unspecific interactions (staining nuclei, mast cells, or keratin). Centering this side specificity and repressing the staining capability of PO was achieved by chemical modification of the respective dye leading to new specific probes for keratin and cytoplasmatic RNA. In conclusion, catalytic cross-linking of heterocyclic 4-N,N-dimethylamino-styryl dyes represents a promising approach for the permanent fluorescent staining of PO in fixed cells and tissues, complementing the CARD technique. In contrast to CARD-related approaches, new substrates are characterized by a broad excitation and emission range of fluorescence and the outstanding spatial resolution of specific fluorescence signaling known so far from their 4-hydroxystyryl analogues. They currently represent the smallest fluorescent substrates of PO. Histochemical and immuno-histochemical applications share several outstanding features: High detection sensitivity, spatial resolution of fluorescence signaling, and photo stability. 4-N,N-dimethylamino-styryl substrates are compatible with their phenol and phenol-ester analogues. Their combination facilitates the trichromatic immuno-histochemical demonstration of three different targets simultaneously at one excitation wavelength in a conventional epi-fluorescence microscope.     

Detection of endogenous and immuno-bound peroxidase — The status Quo in histochemistry

The discovery of synthetic dyes goes back to 1856 and launched the development of the whole chemical and pharmaceutical industry. In life sciences synthetic dyes represent indispensable tools for the microscopic and macroscopic level. Small dyes have the advantage of their easy adaptability to various measuring equipments. By way of structural modification of the chromophore portion, dye labels can be tailored that they absorb and emit light at desired wavelengths ranging from the UV to the near infrared region of the spectrum. Assisted by the development of light measuring techniques and the commercial availability of highly sensitive equipment, today luminescent labels represent most sensitive detection tools in life sciences and dominate over chromogen based techniques. However, for detection of active sites of peroxidase (PO) so far fluorescent labels have been confined to only a few substrates while a broad variety of well-established chromogenic techniques exist. This review covers fluorescent and chromogenic approaches for the permanent detection of immuno-bound and endogenous PO-activity in fixed cells and tissues. Thereby the tailoring of suitable dye labels is additionally challenged by two demands: (1) The applied dye (or its precursor) must act as enzyme substrate specifically and (2) the enzymatic impact must furnish an insoluble dye product from easy soluble starting materials in a very quick reaction. Hence it is not surprising that among PO-substrates (and enzyme substrates generally), dye conjugates represent only an exception while most of these labels represent reactive dyes or suitable precursors. Chromogenic and fluorescent approaches for the permanent labeling of enzymatic sites are compiled. Furthermore, various area-spanning PO-detection principles are discussed ranging from transmission light (TLM) and fluorescence light (FLM) microscopy (chromogenes, flourochromes, fluorescent chromogenes, chromogenes with nonlinear optical properties) to correlated transmission electron microscopy (TEM; photoconversion of specific chromogenic reaction products, electron opaque and/or osmiophilic chromogenic substrates). Also, approaches for reflectance laser microscopy (RLM), polarization microscopy (PM), and correlative TLM, FLM, and multiphoton fluorescence microscopy (MFM) are discussed.     

Novel Oxidative Self-Anchoring Fluorescent Substrates for the Histochemical Localization of Endogenous and Immunobound Peroxidase Activity

Some 2-(2-styryl)-benzothiazole derivatives have been synthesized as novel fluorescent substrates for the localization of peroxidase activity. Excellent localization, high staining sensitivity and exceptionally low background staining were achieved by optimizing the choice of substrate. Multiple step-by-step anchoring of enzymatically-activated individual substrate molecules to surrounding nucleophiles, related to the catalysed reporter deposition (CARD) technique, is discussed. In contrast to tyramine conjugates, as employed in the CARD technique, the separation between reporting and anchoring function is eliminated, thus yielding a new fluorochrome with altered fluorescence properties after enzymatic cross-linking. (E)-2-(2-[4-hydroxyphenyl] vinyl)-3-ethyl-1,3-benzothiazolium iodide has been found to the best substrate so far. This was demonstrated in histochemical applications for the localization of endogenous and immunobound peroxidase activity using fixed cryostat, paraffin or semi-thin Epon sections. The specific final reaction product is efficiently excitable over a wide spectrum from green to violet, providing an outstanding sensitive localization of sites of enzymatic activity with high photo stability. In a comparative study with the Alexa Fluor 546-tyramine conjugate, endogenous and immunobound peroxidase activity was visualized and the results compared using an epi-fluorescence confocal laser scanning microscope. The novel substrate provided an improved specificity and very low background staining whereas the Alexa Fluor-tyramide exhibited a strong overall background staining. FITC-labelled secondary antibodies also yielded very low background staining but the staining was less specific compared with the biotin-based ABC amplification systems labelled with the selected substrate or the Alexa-tyramide. In conclusion, multiple fluorochrome generation close to sites of peroxidase activity, by enzymatic cross-linking of styrene-related substrates, is a promising alternative to the fluorochrome-labelled tyramine ('tyramide') deposition technique.     

Chromogenic substrates for horseradish peroxidase

Two new detection systems for horseradish peroxidase (HRP) have been developed for the staining of membranes used in immunoassays. These systems use dimethyl or diethyl analogues of p-phenylenediamine with 4-chloro-1-naphthol to generate a blue product or 3-methyl-2-benzothiazolinone hydrazone with 4-chloro-1-naphthol to generate a red product. These reagents offer increased sensitivity and lower background staining than currently available chromogenic detection substrates. In addition, the incorporation of these substrates increases the sensitivity of HRP labels to be comparable to that of alkaline phosphatase with the 5-bromo-4-chloro-3-indolyl phosphate + nitro blue tetrazolium substrate.     

Introduction of a novel HRP substrate-Nanogold probe for signal amplification in immunocytochemistry.

Amplification of immunological signals with catalyzed reporter deposition (CARD) allows improved detection of scarce tissue antigens in light and electron microscopy. The technique takes advantage of the oxidation ability of horseradish peroxidase (HRP), in the presence of hydrogen peroxide, to yield the accumulation of one of its specific reporter-tagged substrates. This immunocytochemical approach continues to be improved by the introduction of new reporter molecules tagged to tyramine or to other HRP substrates. In this study we introduced a novel HRP substrate tagged to Nanogold particles. The amplification protocol is based on the application of a specific primary antibody, a biotinylated secondary antibody, streptavidin-HRP, and an HRP substrate coupled to Nanogold, followed by silver intensification. In addition to amplification of immunological signals of high resolution, direct accumulation of Nanogold particles at target sites by enzymatic activity of HRP improves the efficiency of the technique compared to other amplification protocols. Moreover, this approach combines the CARD amplification potentials with the ultrasmall gold probe and the silver intensification method. Immunolabeling obtained by light and electron microscopy, as well as immunodot assay using this new amplification strategy, appear to be highly sensitive, specific, and of enhanced intensity.     

Stabilization of the tetramethylbenzidine (TMB) reaction product: application for retrograde and anterograde tracing, and combination with immunohistochemistry

Tetramethylbenzidine (TMB) as a substrate for horseradish peroxidase (HRP) histochemistry is more sensitive than other chromogens. Its instability in aqueous solutions and ethanol, however, has limited its application. We now report a method for stabilizing TMB by incubation in combinations of diaminobenzidine (DAB)/cobalt (Co2+)/H2O2. The stabilized TMB product was unaffected by long-term exposures to ethanol, neutral buffers, and subsequent immunohistochemical staining procedures. A procedure is recommended for optimal stabilization of TMB that affords a sensitivity for demonstrating retrogradely labeled perikarya comparable to standard TMB histochemistry. The physical characteristics of the reaction product make it suitable for combination with the unlabeled antibody, peroxidase-antiperoxidase (PAP) immunohistochemical staining procedure. This was established by staining retrogradely labeled neurons in the basal forebrain with a monoclonal antibody against choline acetyltransferase. Because the stabilized TMB product exhibited a superior sensitivity over cobalt ion intensification of the DAB-based reaction product (DAB-Co), it offers a distinct advantage over previously described combination procedures.     

Signal amplification in flow cytometry using biotin tyramine.

BACKGROUND: Catalysed reporter deposition (CARD) has been successfully used as a means of signal amplification in solid-phase immunoassays. The procedure relies on the use of horseradish peroxidase (HRP)-conjugated reagents--normally antibodies-in conjunction with substituted phenolic compounds such as biotin tyramine. The HRP catalyses deposition of biotin tyramine around the site of enzyme activity, and streptavidin-HRP can then be added to generate an amplified HRP signal. The possibility of using this technique for solution-phase amplifications has been suggested but not yet demonstrated. METHODS: This paper describes the application of CARD to signal enhancement in flow cytometry. The specific examples described here are those of anti-human CD4 and anti-human CD36 antibodies binding to either human lymphocytes or mixed mononuclear cells. RESULTS: Optimum biotin tyramine concentrations were evaluated, and a fivefold increase in signal was observed over standard detection of the anti-human CD4 antibody with anti-mouse-fluorescein isothiocyanate (FITC). In the example using the anti-CD36 antibody, the biotin tyramine treatment was repeated, resulting in an additional 2.5-fold signal amplification. CONCLUSIONS: The technique described in this report provides a method of amplifying the signals achieved by standard flow cytometry detection reagents.     

Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains.

A procedure is described for intensifying histochemical reactions by amplification of biotinylated sites. This is achieved by deposition of biotinylated tyramine on the tissue through the enzymatic action of horseradish peroxidase (HRP). The amplified biotin sites are subsequently visualized by binding them to avidin, to which a marker is attached. This amplification greatly increases the sensitivity of staining procedures that employ HRP (and/or biotin) in tissue. For neuroanatomical pathway tracing methods, the procedure greatly increases the detectability of the injected tracer. For lectin histochemistry and immunohistochemistry, the amplification requires that the lectin or primary antibody be greatly diluted. This dilution results in less background staining and yet strong signals are produced even when very dilute reagents are used. Alternatively, the amplification permits much shorter incubations in primary antibodies when dilutions are used that would ordinarily be used with conventional bridge techniques. The procedure is also useful for amplifying very weak signals, such as those of immunoreactions in glutaraldehyde-fixed tissue. The amplification procedure, together with the availability of avidin probes labeled with fluorochromes, colloidal gold, or enzyme systems other than HRP, provides a means of greatly increasing the versatility of a variety of histochemical reactions, including those for detecting in situ hybridization probes, in addition to increasing the sensitivity of the reactions.     

Glucose oxidase as label in histological immunoassays with enzyme-amplification in a two-step technique: coimmobilized horseradish peroxidase as secondary system enzyme for chromogen oxidation

A sensitive staining procedure for glucose oxidase (GOD) as marker in immunohistology is described. The cytochemical procedure involves a two-step enzyme method in which GOD and horseradish peroxidase (HRP) are coimmobilized onto the same cellular sites by immunological bridging or by the principle of avidin-biotin interaction. In this coupled enzyme technique, H2O2 generated during GOD reaction is the substrate for HRP and is utilized for the oxidation of chromogens such as 3,3'-diaminobenzidine or 3-amino-9-ethylcarbazole. Due to the immobilization of the capture enzyme HRP in close proximity to the marker enzyme (GOD), more intense and specific staining is produced than can be obtained with soluble HRP as coupling enzyme in the substrate medium. Indirect antibody labelled and antibody bridge techniques including the avidin (streptavidin)-biotin principle have proven the usefulness of this GOD labelling procedure for antigen localization in paraffin sections. Antigens such as IgA in tonsil, alpha-fetoprotein in liver and tissue polypeptide antigen in mammary gland served as models. The immobilized two-step enzyme procedures have the same order of sensitivity and specificity as comparable immunoperoxidase methods. The coupled GOD-HRP principle can be superior to conventional immunoperoxidase labelling for the localization of biomolecules in tissue preparations rich in endogenous peroxidase activities.     

Immunogold signal amplification: Application of the CARD approach to electron microscopy.

Catalyzed reporter deposition (CARD) is a technique that allows amplification of routine immunolabeling in light microscopy. This procedure takes advantage of the horseradish peroxidase (HRP) from an HRP-avidin complex to catalyze the accumulation of reporter-conjugated tyramine (a phenolic compound) onto a surface displaying biotinylated antigen-antibody complexes. The large amount of labeled tyramine deposited allows the detection of an antigenic site with multiple reporter molecules. In this study we modified this amplification protocol to combine it with the immunogold technique for the ultrastructural localization of antigens in electron microscopy. We constructed various tyramide conjugates that permit the combination of this amplification method with a particulate colloidal gold marker. The new probes yield results of high specificity and enhanced intensity. Assessment of the level of resolution of the labeling has demonstrated that, in spite of the amplification, the resolution remains very good. Therefore, once associated, the immunogold and the CARD techniques lead to specific, high-resolution, sensitive and amplified signals that exhibit the advantages of both approaches.(J Histochem Cytochem 47:421-429, 1999)     

Glucose oxidase as label in histological immunoassays with enzyme-amplification in a two-step technique: coimmobilized horseradish peroxidase as secondary system enzyme for chromogen oxidation

Summary A sensitive staining procedure for glucose oxidase (GOD) as marker in immunohistology is described. The cytochemical procedure involves a two-step enzyme method in which GOD and horseradish peroxidase (HRP) are coimmobilized onto the same cellular sites by immunological bridging or by the principle of avidin-biotin interaction. In this coupled enzyme technique, H₂O₂ generated during GOD reaction is the substrate for HRP and is utilized for the oxidation of chromogens such as 3,3-diaminobenzidine or 3-amino-9-ethylcarbazole. Due to the immobilization of the capture enzyme HRP in close proximity to the marker enzyme (GOD), more intense and specific staining is produced than can be obtained with soluble HRP as coupling enzyme in the substrate medium. Indirect antibody labelled and antibody bridge techniques including the avidin (streptavidin)-biotin principle have proven the usefulness of this GOD labelling procedure for antigen localization in paraffin sections. Antigens such as IgA in tonsil, alpha-feroprotein in liver and tissue polypeptide antigen in mainmary gland served as models. The immobilized twostep enzyme procedures have the same order of sensitivity and specificity as comparable immunoperoxidase methods. The coupled GOD-HRP principle can be superior to conventional immunoperoxidase labelling for the localization of biomolecules in tissue preparations rich in endogenous peroxidase activities.     

In situ identification of cyanobacteria with horseradish peroxidase-labeled, rRNA-targeted oligonucleotide probes

Individual cyanobacterial cells are normally identified in environmental samples only on the basis of their pigmentation and morphology. However, these criteria are often insufficient for the differentiation of species. Here, a whole-cell hybridization technique is presented that uses horseradish peroxidase (HRP)-labeled, rRNA-targeted oligonucleotides for in situ identification of cyanobacteria. This indirect method, in which the probe-conferred enzyme has to be visualized in an additional step, was necessary since fluorescently monolabeled oligonucleotides were insufficient to overstain the autofluorescence of the target cells. Initially, a nonfluorescent detection assay was developed and successfully applied to cyanobacterial mats. Later, it was demonstrated that tyramide signal amplification (TSA) resulted in fluorescent signals far above the level of autofluorescence. Furthermore, TSA-based detection of HRP was more sensitive than that based on nonfluorescent substrates. Critical points of the assay, such as cell fixation and permeabilization, specificity, and sensitivity, were systematically investigated by using four oligonucleotides newly designed to target groups of cyanobacteria.     

Towards Q-PCR of pathogenic bacteria with improved electrochemical double-tagged genosensing detection

A very sensitive assay for the rapid detection of pathogenic bacteria based on electrochemical genosensing has been designed. The assay was performed by the PCR specific amplification of the eaeA gene, related with the pathogenic activity of Escherichia coli O157:H7. The efficiency and selectivity of the selected primers were firstly studied by using standard Quantitative PCR (Q-PCR) based on TaqMan fluorescent strategy. The bacteria amplicon was detected by using two different electrochemical genosensing strategies, a highly selective biosensor based on a bulk-modified avidin biocomposite (Av-GEB) and a highly sensitive magneto sensor (m-GEC). The electrochemical detection was achieved in both cases by the enzyme marker HRP. The assay showed to be very sensitive, being able to detect 4.5 ng microl(-1) and 0.45 ng microl(-1) of the original bacterial genome after only 10 cycles of PCR amplification, when the first and the second strategies were used, respectively. Moreover, the electrochemical strategies for the detection of the amplicon showed to be more sensitive compared with Q-PCR strategies based on fluorescent labels such as TaqMan probes.     

Chemiluminescent-based methods to detect subpicomole levels of c-type cytochromes

A variety of luminol-based substrates and either an autoradiographic film or a charge-coupled device (CCD) imaging system were used for chemiluminescence detection of c-type cytochromes. The Pierce Femto peroxidase substrate was at least 10 times more sensitive when using film than the highly cited 3,3('),5,5(')-tetramethylbenzidine (benzidine derivative) staining method and 50 times more sensitive when using a CCD imaging system. Film or CCD imaging result in highly sensitive and quantitative signals. The quantitative detection of c-type cytochromes from single colonies or from less than 1ml of a bacterial culture is possible.     

Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry

Summary Various chromogen protocols for visualizing peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry were compared with respect to their sensitivity. They were tested on tissue sections of human skeletal muscle and in an antigen spot test using antibodies against slow skeletal muscle myosin. The chromogens included 3-amino-9-ethylcarbazole (AEC), 3, 3-diaminobenzidine (DAB),p-phenylenediamine-pyrocatechol (PPD-PC) and 4-chloro-1-naphthol (CN) in peroxidase histochemistry, and 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium salt (BCIP-NBT), BCIP-tetra nitro blue tetrazolium salt (TNBT) and various combinations of substituted naphthol phosphate-diazonium salt in alkaline phosphatase histochemistry. DAB, CN, and PPD-PC were also employed with imidazole and DAB in addition to Co²⁺ and Ni²⁺ ions. The results indicate that DAB-imidazole and DAB-Co²⁺ and Ni²⁺ ions are the most sensitive chromogen protocols for visualizing peroxidase activity. Although no large differences were found between the various chromogen protocols for visualizing alkaline phosphatase activity, the protocol BCIP-TNBT is especially recommended. Furthermore, the various chromogen protocols were evaluated as to stability of chromogen solutions and final precipitates, background staining, localization properties, and enhancement of enzyme activity.     

Multienzyme-nanoparticles amplification for sensitive virus genotyping in microfluidic microbeads array using Au nanoparticle probes and quantum dots as labels

A novel microfluidic device with microbeads array was developed and sensitive genotyping of human papillomavirus was demonstrated using a multiple-enzyme labeled oligonucleotide-Au nanoparticle bioconjugate as the detection tool. This method utilizes microbeads as sensing platform that was functionalized with the capture probes and modified electron rich proteins, and uses the horseradish peroxidase (HRP)-functionalized gold nanoparticles as label with a secondary DNA probe. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. Through "sandwich" hybridization, the enzyme-functionalized Au nanoparticles labels were brought close to the surface of microbeads. The oxidation of biotin-tyramine by hydrogen peroxide resulted in the deposition of multiple biotin moieties onto the surface of beads. This deposition is markedly increased in the presence of immobilized electron rich proteins. Streptavidin-labeled quantum dots were then allowed to bind to the deposited biotin moieties and displayed the signal. Enhanced detection sensitivity was achieved where the large surface area of Au nanoparticle carriers increased the amount HRP bound per sandwiched hybridization. The on-chip genotyping method could discriminate as low as 1fmol/L (10zmol/chip, SNR>3) synthesized HPV oligonucleotides DNA. The chip-based signal enhancement of the amplified assay resulted in 1000 times higher sensitivity than that of off-chip test. In addition, this on-chip format could discriminate and genotype 10copies/μL HPV genomic DNA using the PCR products. These results demonstrated that this on-chip approach can achieve highly sensitive detection and genotyping of target DNA and can be further developed for detection of disease-related biomolecules at the lowest level at their earliest incidence.     

Horseradish peroxidase-functionalized gold nanoparticle label for amplified immunoanalysis based on gold nanoparticles/carbon nanotubes hybrids modified biosensor

This paper describes the combination of electrochemical immunosensor using gold nanoparticles (GNPs)/carbon nanotubes (CNTs) hybrids platform with horseradish peroxidase (HRP)-functionalized gold nanoparticle label for the sensitive detection of human IgG (HIgG) as a model protein. The GNPs/CNTs nanohybrids covered on the glass carbon electrode (GCE) constructed an effective antibody immobilization matrix and made the immobilized biomolecules hold high stability and bioactivity. Enhanced sensitivity was obtained by using bioconjugates featuring HRP labels and secondary antibodies (Ab₂) linked to GNPs at high HRP/Ab₂ molar ratio. The approach provided a linear response range between 0.125 and 80 ng/mL with a detection limit of 40 pg/mL. The immunosensor showed good precision, acceptable stability and reproducibility and could be used for the detection of HIgG in real samples, which provided a potential alternative tool for the detection of protein in clinical laboratory.     

A reliable method combining horseradish peroxidase histochemistry with immuno-beta-galactosidase staining

A sensitive combination of horseradish peroxidase (HRP) tracing and immunohistochemistry was used by Rye et al. [J Histochem Cytochem (1984) 32:1145] in a search for the origins of neurotransmitter- and neuromodulator-containing nerve fibers in brain. In this combination, peroxidase as a marker in immunohistochemistry was thought to yield a homogeneous brown immunoreaction product of diaminobenzidine, different from the black granular reaction product of retrogradely transported HRP, which is visualized by the tetramethylbenzidine (TMB) reaction and subsequent stabilization. A neuron that exhibits both kinds of reaction products in its cytoplasm in sections subjected to combination staining is referred to as a double-labeled cell. With a combined HRP and corticotropin-releasing factor (CRF) immunoperoxidase-antiperoxidase (PAP) method, the first set of experiments showed "false" double-labeled cells in the pyramidal cell layer of rat cerebral cortex, but only rarely in the subcortical areas, possibly because of the use of one enzyme system in two different histochemical procedures. This limitation of the double-staining technique prompted us to demonstrate an alternate combination of HRP tracing and immunohistochemistry in the second set of experiments by employing two previously described independent enzyme systems: HRP as a retrograde tracer and beta-galactosidase as a marker for immunohistochemical demonstration of CRF. A homogeneous blue reaction product indicated immuno-beta-galactosidase staining, and a granular black or brown reaction product labeled retrogradely transported HRP in double-labeled cells in subcortical regions. Neither double labeling nor "false" double labeling was seen in pyramidal cells of cerebral cortex. These findings suggest that application of two independent enzyme systems in a combined HRP and immunohistochemical method may be useful for investigating in origins of peptidergic fibers in brain when the combination of HRP histochemistry and the PAP method appears to be inappropriate.