Binding Of Horseradish Peroxidase Research Articles

A Simple Biosensor Based on Streptavidin-HRP for the Detection of Bacteria Exploiting HRPs Molecular Surface Properties

Bacterial contamination of water and food sources is still a major source of diseases. Early detection of potential pathogens is key to prevent their spreading and severe health risks. Here, we describe a fast, low-cost detection assay based on horseradish peroxidase (HRP) conjugated to streptavidin for the direct identification of bacteria. Streptavidin can bind to bacterial cells due to its high affinity for biotin, a natural component of microbial cell surfaces. Upon binding to bacteria, the HRP converts a chromogenic substrate, resulting in a visible color change. In the present study, we evaluated different detection platforms regarding their compatibility with the detection principle. To reduce background signals and increase the sensitivity of HRP-based assays, the binding of HRP to surfaces and biomolecules was intensively investigated. The final assay successfully detected the most relevant bacterial strains in drinking water, such as Escherichia coli, Klebsiella pneumonia, and Enterobacter cloacae.

Immobilization of Horseradish Peroxidase on Magnetite-Alginate Beads to Enable Effective Strong Binding and Enzyme Recycling during Anthraquinone Dyes' Degradation.

The aim of this study was to investigate covalent immobilization of horseradish peroxidase (HRP) on magnetic nanoparticles (Mag) encapsulated in calcium alginate beads (MABs) for color degradation, combining easy and fast removal of biocatalyst from the reaction mixture due to its magnetic properties and strong binding due to surface alginate functional groups. MABs obtained by extrusion techniques were analyzed by optical microscopy, FEG-SEM and characterized regarding mechanical properties, magnetization and HRP binding. HRP with initial concentration of 10 mg/gcarrier was successfully covalently bonded on MABs (diameter ~1 mm, magnetite/alginate ratio 1:4), with protein loading of 8.9 mg/gcarrier, immobilization yield 96.9% and activity 32.8 U/g. Immobilized HRP on MABs (HRP-MABs) was then used to catalyze degradation of two anthraquinonic dyes, Acid Blue 225 (AB225) and Acid Violet 109 (AV109), as models for wastewater pollutants. HRP-MABs decolorized 77.3% and 76.1% of AV109 and AB225, respectively after 15 min under optimal conditions (0.097 mM H2O2, 200 mg of HRP-MABs (8.9 mg/gcarrier), 0.08 and 0.1 g/mg beads/dye ratio for AV109 and AB225, respectively). Biocatalyst was used for 7 repeated cycles retaining 75% and 51% of initial activity for AB225 and AV109, respectively, showing potential for use in large scale applications for colored wastewater treatment.

Molecularly imprinted colloidal array with multi-boronic acid sites for glycoprotein detection under neutral pH

Glycoproteins play vital roles in living organisms and often serve as biomarkers for some disease. However, due to the low content of glycoprotein in biological fluids, selective detection of glycoproteins is still a challenging issue that needs to be addressed. In this study, molecularly imprinted colloidal array with multi-boronic acid sites for glycoprotein detection under physiological pH was proposed. Monodispersed glycoprotein imprinted particles (SiO2@PEI/MIPs) was first prepared based on surface imprinting strategy using horseradish peroxidase (HRP) as template, and polyethyleneimine (PEI) was used to increase the number of boronic acid groups. The binding experiment indicated that the SiO2@PEI/MIPs hold satisfactory adsorption capacity (1.41 μmol/g), rapid adsorption rate (40 min) and preferable selectivity toward HRP. Then the SiO2@PEI/MIPs was assembled into close-packed colloidal array to construct a label free optical sensor (denoted as GICA). Benefiting from the high ordered photonic crystal structure, binding of HRP onto the GICA could be directly readout from the changes in structure color and diffracted wavelength. The structure color of the GICA changed from bright blue to yellow with the diffraction wavelength red shifted 59 nm when the HRP concentration increased from 2.5 to 15 μmol/L. Importantly, the GICA was capable of detecting HRP from human serum samples. All those results indicated the potential of the GICA for naked-eye detection of glycoprotein.

Molecularly imprinted polyaniline for detection of horseradish peroxidase.

A new facile and fast approach to the synthesis of polyaniline (PANi) molecularly imprinted polymers (MIPs) based on aniline oxidative chemical polymerization was proposed for protein recognition. For the first time, a surface imprinting strategy was implemented for the synthesis of PANi MIPs on the inner surface of soft glass polycapillaries (PC) with a large (2237) number of individual microcapillaries. Two different PANi layers-(i) PANi film and (ii) protein imprinted PANi nanowires-were synthesized sequentially. Uniform and highly stable PANi film was synthesized by oxidative polymerization at pH< 1. The synthesis of PANi MIPs on the PANi film pre-coated surface improved the reproducibility of PANi MIP formation. PANi MIP nanowires were synthesized at "mild" conditions (pH > 4.5) to preserve the protein template activity. The binding of horseradish peroxidase (HRP) molecules on the PANi MIP selective sites was confirmed by photometry (TMB chromogenic reaction), SEM images, and FTIR spectroscopy. The developed PANi MIPs enable HRP determination with a limit of detection (LOD) as low as 1.00 and 0.07ngmL-1 on the glass slips and PC, respectively. The PANi MIPs are characterized by high stability; they are reversible and selective to HRP. The proposed approach allows PANi MIPs to be obtained for proteins on different supports and to create new materials for separation and sensing. Graphical abstract.

A close-packed imprinted colloidal array for naked-eye detection of glycoproteins under physiological pH

According to the combination of colloidal crystals and molecular imprinting techniques, a novel close-packed imprinted colloidal array (CPICA) for naked-eye horseradish peroxidase (HRP) detection at physiological pH was proposed. The CPICA was fabricated by self-assemble of monodispersed HRP imprinted particles. The HRP imprinted particles were prepared based on surface imprinting technique and immobilized template strategy using 2,4-difluoro-3-formylphenylboronic acid (DFFPBA) as functional monomer which allowed the material binding of HRP at physiological pH (denoted as SiO2@DFFPBA/MIPs). The adsorption capacity of the SiO2@DFFPBA/MIPs for HRP was 1.16 μmol/g, and reached saturated adsorption within 25 min. The limit of detection (LOD) of the CPICA was 3.0 × 10−13 mol/mL. In addition, the adsorption of HRP on the CPICA could be directly transferred into visible color changes and readable optical signals through the reflection peak shifts. The structure color of the CPICA changed from brilliant blue to dark red with an maximum red shift of 87 nm when the HRP concentration increased from 2.5 to 20.0 μmol/L. Moreover, the CPICA could be used to detect HRP from human serum sample, which demonstrated the promising application prospects in colorimetric sensors.

Detection of the Malaria causing Plasmodium Parasite in Saliva from Infected Patients using Topoisomerase I Activity as a Biomarker

Malaria is among the major threats to global health with the main burden of disease being in rural areas of developing countries where accurate diagnosis based on non-invasive samples is in high demand. We here present a novel molecular assay for detection of malaria parasites based on technology that may be adapted for low-resource settings. Moreover, we demonstrate the exploitation of this assay for detection of malaria in saliva. The setup relies on pump-free microfluidics enabled extraction combined with a DNA sensor substrate that is converted to a single-stranded DNA circle specifically by topoisomerase I expressed by the malaria causing Plasmodium parasite. Subsequent rolling circle amplification of the generated DNA circle in the presence of biotin conjugated deoxynucleotides resulted in long tandem repeat products that was visualized colorimetrically upon binding of horse radish peroxidase (HRP) and addition of 3,3′,5,5′-Tetramethylbenzidine that was converted to a blue colored product by HRP. The assay was directly quantitative, specific for Plasmodium parasites, and allowed detection of Plasmodium infection in a single drop of saliva from 35 out of 35 infected individuals tested. The results could be determined directly by the naked eye and documented by quantifying the color intensity using a standard paper scanner.

Concanavalin A photocross-linked affinity cryogels for the purification of horseradish peroxidase

The present study describes an easy and efficient procedure for the purification of horseradish peroxidase from horseradish roots. For this purpose, supermacroporous cryogels having Concanavalin A were prepared by photosensitive cross-linking polymerization. Horseradish peroxidase binding and elution from the prepared cryogels were carried out changing various parameters such as initial peroxidase concentration and pH. The best binding performance was obtained at pH 7.0. The maximum horseradish peroxidase binding of the cryogels was found to be 3.85 mg g−1cryogel. Horseradish peroxidase purification from crude extract resulted in 115.1-fold. SDS-PAGE analysis and circular dichroism measurements indicated that the horseradish peroxidase purification from horseradish roots was successfully carried out.

Ultrasensitive Photoelectrochemical Biosensing of Cell Surface N-Glycan Expression Based on the Enhancement of Nanogold-Assembled Mesoporous Silica Amplified by Graphene Quantum Dots and Hybridization Chain Reaction.

An ultrasensitive photoelectrochemical (PEC) biosensor for N-glycan expression based on the enhancement of nanogold-assembled mesoporous silica nanoparticles (GMSNs) was fabricated, which also combined with multibranched hybridization chain reaction (mHCR) and graphene quantum dots (GQDs). In this work, the localized surface plasmon resonance, mHCR and GQDs-induced signal amplification strategies were integrated exquisitely and applied sufficiently. In the fabrication, after porous ZnO spheres immobilized on the Au nanorod-modified paper working electrode were sensitized by CdTe QDs, the GMSNs were assembled on the CdTe QDs. Then the photocurrent efficiency was improved by the sensitization of the CdTe QDs and the localized surface plasmon resonance of GMSNs. Successively, the products of mHCR with multiple biotins for multiple horseradish peroxidase binding and multiple branched arms for capturing the target cells were attached on the as-prepared electrode. The chemiluminescent (CL) emission with the aid of horseradish peroxidase served as an inner light source to excite photoactive materials for simplifying the instrument. Furthermore, the aptamer could capture the cancer cells by its highly efficient cell recognition ability, which avoided the conventional routing cell counting procedures. Meanwhile, the GQDs served as the signal amplication strategy, which was exerted in the process of N-glycan evaluation because the competitive absorption of exciting light and consumption of H2O2 served as the electron donor of the PEC system and the oxidant of the luminol-based CL system. This judiciously engineered biosensor offered a promising platform for the exploration of N-glycan-based physiological processes.

Enhancing coupled enzymatic activity by conjugating one enzyme to a nanoparticle.

Enzymes have long been a prime research target for the commercial production of commodity and specialty chemicals, design of sensing devices, and the development of therapeutics and new chemical processes. Industrial applications for enzymes can potentially be enhanced by enzyme immobilization which often allows for increased enzyme stability, facile product purification, and minimized substrate diffusion times in multienzymatic cascades, but this is usually at the cost of a significant decrease in catalytic rates. Recently, enzyme immobilization has been advanced by the discovery that nanoparticle surfaces are frequently able to enhance the activity of the bound enzyme. Here we extend this observation to a multienzymatic coupled system using semiconductor quantum dots (QDs) as a model nanoparticle material and the prototypical enzyme pair of glucose oxidase (GOX) and horseradish peroxidase (HRP). We first demonstrate that HRP binding to QDs has a significant beneficial effect on enzymatic activity, producing a >2-fold improvement in kcat. We argue that this enhancement is due to affinity of the QD surface for the substrate. Furthermore, we demonstrate that when the ratio of GOX to HRP is adjusted to allow HRP to be the rate-limiting step of the pathway, the QD-induced rate enhancement of HRP can be maintained in a multi-enzyme cascade. Kinetic analysis shows that the underlying processes can be simulated numerically and provide insight into the governing mechanisms. The potential of nanoparticle-based catalytic enhancement is then discussed in the context of multienzyme cascades and synthetic biology.

Controlled chemical modification of the internal surface of photonic crystal fibers for application as biosensitive elements

Photonic crystal fibers (PCF) are one of the most promising materials for creation of constructive elements for bio-, drug and contaminant sensing based on unique optical properties of the PCF as effective nanosized optical signal collectors. In order to provide efficient and controllable binding of biomolecules, the internal surface of glass hollow core photonic crystal fibers (HC-PCF) has been chemically modified with silanol groups and functionalized with (3-aminopropyl) triethoxysilane (APTES). The shift of local maxima in the HC-PCF transmission spectrum has been selected as a signal for estimating the amount of silanol groups on the HC-PCF inner surface. The relationship between amount of silanol groups on the HC-PCF inner surface and efficiency of following APTES functionalization has been evaluated. Covalent binding of horseradish peroxidase (chosen as a model protein) on functionalized PCF inner surface has been performed successively, thus verifying the possibility of creating a biosensitive element.

A β-cyclodextrin–chitosan complex as the immobilization matrix for horseradish peroxidase and its application for the removal of azo dyes from textile effluent

The aim of this study was to develop a system based on the use of immobilized horseradish peroxidase (HRP) for the removal of color due to azo dyes from a textile effluent. The maximum binding of HRP on a β-cyclodextrin (CD)–chitosan complex was achieved at pH 8.0. Oxidative removal of azo dyes from effluent by water-soluble and β-CD–chitosan–HRP inclusion complexes was monitored by HPLC analysis. The crosslinked peroxidase preparation made with a β-CD–chitosan complex was more active, effective and stable than the un-crosslinked and the water-soluble enzyme preparations. The maximum dye oxidation from the diluted textile effluent was observed in the presence of 0.6 mM H2O2 and 0.2 U ml−1 at pH 8.0 and at 40 °C for 2 h. A batch process with HRP was also performed, whereby complete dye removal from the effluent was obtained with a crosslinked β-CD–chitosan complex within 120 min. On the other hand, a β-CD–chitosan–HRP-based continuous bed reactor was found to be efficient for complete removal of the dye from the effluent during 15 days of operation. The reuse of the crosslinked enzyme preparations proved to be more effective in the decolorization of textile effluent than the un-crosslinked enzyme preparations. Based on a comet assay the genotoxicity associated the colored effluent was completely lost after HRP oxidation of the dye, showing the dye metabolites are not toxic.

A novel hydrogen peroxide biosensor based on the specific binding of horseradish peroxidase with polymeric thiophene-3-boronic acid monolayer in hydrophilic room temperature ionic liquid

In the present work, we have developed a novel hydrogen peroxide (H 2O 2) biosensor based on electropolymerization of thiophene-3-boronic acid film (TBA) in ionic liquid via glucosidic bond with glycoprotein horseradish peroxidase (HRP). A thin film of poly (thiophene-3-boronic acid) (PTBA) has been synthesized by electropolymerization using room temperature ionic liquids (RTIL) as the growth medium and the supporting electrolyte. Horseradish peroxidase (HRP) in this study shows affinity interaction with the formed PTBA thin film. With the aid of hydroquinone (H 2Q) mediator, the biosensor has a fast response of less than 7 s with linear range of 5.0 × 10 −6 to 1.75 × 10 −4 M and a detection limit of 0.5 μM. The developed biosensor exhibits fast response, a low detection limit, high stability and very good reproducibility.

Immobilisation of horseradish peroxidase on Eupergit ®C for the enzymatic elimination of phenol

In this study, three different approaches for the covalent immobilisation of the horseradish peroxidase (HRP) onto epoxy-activated acrylic polymers (Eupergit ®C) were explored for the first time, direct HRP binding to the polymers via their oxirane groups, HRP binding to the polymers via a spacer made from adipic dihydrazide, and HRP binding to hydrazido polymer surfaces through the enzyme carbohydrate moiety previously modified by periodate oxidation. The periodate-mediated covalent immobilisation of the HRP on hydrazido Eupergit ®C was found to be the most effective method for the preparation of biocatalysts. In this case, a maximum value of the immobilised enzyme activity of 127 U/g support was found using an enzyme loading on the support of 35.2 mg/g support. The free and the immobilised HRP were used to study the elimination of phenol in two batch reactors. As expected, the activity of the immobilised enzyme was lower than the activity of the free enzyme. Around 85% of enzyme activity is lost during the immobilisation. However, the reaction using immobilised enzyme showed that it was possible to reach high degrees of phenol removal (around 50%) using about one hundredth of the enzyme used in the soluble form.

Dendrobium findleyanum agglutinin: production, localization, anti-fungal activity and gene characterization

The recently reported Dendrobium findleyanum agglutinin (DFA) was identified and determined in different parts of D. findleyanum pseudobulbs by using Western blot analysis, LC-MS/MS, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and histochemical procedure. Western blot analysis of crude protein extract with horseradish peroxidase (HRP), a mannose-rich glycoprotein, showed only one band at 14.5 kDa, which had the same molecular mass as DFA. This band was a major band when the membrane was stained with Coomassie Brilliant Blue. The protein profiles from SDS-PAGE showed higher band intensity of the 14.5 kDa mannose-binding protein in nearly mature and mature stages, compared to very young and young stages of the orchid. In addition, the band intensity was to a great extent different between the swollen and the non-swollen internode of the pseudobulb. Using LC-MS/MS, the sequence tags of the 14.5-kDa protein bands from the node, swollen internode and non-swollen internode revealed that the protein was DFA. Histochemical procedure in the transverse section of the pseudobulbs demonstrated major HRP binding sites, which reflected the location of DFA, in periphery of parenchymal cells. The purified DFA showed anti-fungal activity against Alternaria alternata and Collectotrichum sp. Using reverse transcription polymerase chain reaction and DNA sequencing, the deduced amino acid sequence of the DFA precursor revealed 94% homology with a lectin precursor from D. officinale. N-terminal sequencing demonstrated the processing site between residues 24 and 25 of the DFA precursor.

Plasma immersion ion implantation treatment of polyethylene for enhanced binding of active horseradish peroxidase

Robust attachment of active proteins to synthetic surfaces underpins the development of biosensors and protein arrays. This paper presents the results of experiments in which energetic ions, extracted from an inductively coupled nitrogen plasma, are used to modify the surface of ultrahigh molecular weight polyethylene (UHMWPE). The ability of the surface to bind active horseradish peroxidase (HRP) is significantly enhanced by the plasma treatment. The amide signal in infrared spectroscopy indicates an increased quantity of surface-attached protein on the modified surface. The activity of the bound HRP remains high compared with that of protein attached to the untreated surface, after repeated washing in buffer solution. Although Tween 20 was an effective blocking agent for the unmodified polyethylene surface, binding of HRP to the modified surface is not inhibited by its presence. We propose that the treatment produces new binding sites on the surface and that the function of the HRP is retained because the treated surface is substantially more hydrophilic.

Photochemical activation of a polycarbonate surface for covalent immobilization of a protein ligand

Polycarbonate—a thermostable polymer is activated by a simple and rapid method using a photolinker, 1-fluoro-2-nitro-4-azidobenzene (FNAB) for covalent immobilization of a biomolecule. Horseradish peroxidase (HRP) is used as a model enzyme to check the efficacy of the activated surface. HRP is immobilized on the activated polycarbonate surface without addition of any reagent or catalyst and is found to give 2–2.5-fold increase in absorbance with the substrate as compared to the directly adsorbed enzyme. Photochemical attachment of FNAB to the PC surface is confirmed by X-ray photoelectron spectroscopy (XPS), which shows the presence of nitrogen and fluorine in the ratio of 2:1 in the activated polycarbonate. Disappearance of fluorine peak in the XP spectra of PC bound enzyme further confirms the covalent binding of HRP, through displacement of fluorine moiety of the activated PC by the amino group of the protein. Optimized concentration of the photolinker is found as 6 μmol of FNAB per well and time of photo irradiation is 8 min for activation of a PCR polycarbonate plate. PC bound HRP has shown enhanced thermal and storage stability. Kinetic studies of the immobilized HRP shows improved catalytic activity. The potential application of activated polycarbonate surface includes immobilization of biomolecules for biosensors, immunoassays, and protein and DNA micro-arrays. Due to the stability of the polycarbonate at high temperature, the activated polycarbonate has an advantage for immobilization of thermostable biomolecule such as thermostable enzyme for reaction at elevated temperature.

Horseradish peroxidase immobilized through its carboxylic groups onto a polyacrylonitrile membrane: comparison of enzyme performances with inorganic beaded supports.

A hydrophilic polyacrylonitrile (PAN) flat sheet membrane was aminated (8.5 micromol of NH2/mg of dry support) for covalent binding of horseradish peroxidase (HRP), mediated by the soluble carbodiimide l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC). Silica microbeads derivatized by silanization, to yield an aminated support, and commercial aminated glass microbeads were also coupled to HRP with EDC or activated with glutaraldehyde. The immobilized enzyme activities were determined in a batch enzyme reactor with an external loop, the highest specific immobilized HRP activity being obtained on the glass support (55.8 U/mg of protein). Continuous operational stability studies showed that hydrophilic PAN membrane led to the highest retention of HRP activity after an overall period of 35 h, with a normalized productivity of 59.5 micromol of H2O2 reduced/(h x Uimmob HRP).

A nonclassical estrogen membrane receptor triggers rapid differential actions in the endocrine pancreas.

Glucose homeostasis in blood is mainly maintained by insulin released from beta-cells and glucagon released from alpha-cells, both integrated within the pancreatic islet of Langerhans. The secretory processes in both types of cells are triggered by a rise in intracellular calcium concentration ([Ca2+](i)). In this study, rapid effects of the natural hormone E2 on [Ca2+](i) were studied in both types of cells within intact islets using laser scanning confocal microscopy. alpha- And beta-cells showed opposite [Ca2+](i) responses when stimulated with physiological concentrations of 17beta-E2. Although the estrogen produced an increase in the frequency of glucose-induced [Ca2+](i) oscillations in insulin-releasing beta-cells, it prevented the low glucose-induced [Ca2+](i) oscillations in glucagon-releasing alpha-cells. The effects of 17beta-E2 on alpha-cells were mimicked by the cGMP permeable analog 8bromo-cGMP and blocked by the cGMP-dependent protein kinase (PKG) inhibitor KT5823. Evidence indicated that these were membrane actions mediated by a nonclassical ER. Both effects were rapid in onset and were reproduced by 17beta-E2 linked to horseradish peroxidase, a cell-impermeable molecule. Furthermore, these actions were not blocked by the specific ER blocker ICI 182,780. Competition studies performed with 17beta-E2 linked to horseradish peroxidase binding in alpha-cells supported the idea that the membrane receptor involved is neither ERalpha nor ERbeta. Additionally, the binding site was shared by the neurotransmitters epinephrine, norepinephrine, and dopamine and had the same pharmacological profile as the receptor previously described for beta-cells. Therefore, rapid estrogen actions in islet cells are initiated by a nonclassical estrogen membrane receptor.

An ultrathin platinum film sensor to measure biomolecular binding

A sensitive conductimetric immunosensor has been demonstrated based on an ultrathin platinum film on an oxidized silicon base. The film is about 25 Å thick and is seen to consist of a discontinuous layer with channels 20–30 Å wide. Monoclonal antibodies were bound to the sensor surface using conventional biosensor chemistry. Impedance at fixed frequencies across the film was used to track modification and binding at the surface. Impedance increased 55% at 20 Hz during the activation of the surface with anti-alkaline phosphatase (anti-AP). Binding of alkaline phosphatase (AP) to the prepared surface results in a further increase of 12%. p-Nitrophenyl phosphate hydrolysis confirmed binding and activity of the AP. About 40 amol AP were bound on the 0.5 cm 2 electrode. Non-specific binding of horseradish peroxidase caused an impedance change <6%. Control experiments showed small impedance changes and trace enzyme activity. Since the mechanism of electrical conduction of the thin film was not established, modeling of thin-film response was used to distinguish between redox processes, capacitance and tunneling mechanisms. The data fit well with the diffusion distributed elements (DE) model as well as a transmission line distribution element (DX) model. The first model, DE, is distributed elements for diffusion. The second DX model represents a transmission line. The sensors behave in a distributed network or like a transmission line.

Optical detection of pesticides and drugs based on chemiluminescence–fluorescence assays

The paper illustrates two different concepts for monitoring pesticides and drugs. In the first approach double stranded deoxyribonucleic acid (dsDNA) was used as the sensing material, which intercalated drugs with high affinity. The intercalation of drugs such as Chlorpromazine (CP), Clomipramine (CM) and Imipramine (IP) were investigated. The assay was based on competition between the drug molecule and a fluorescent dye (ToPro-3) for intercalation with dsDNA. The ToPro3 was excited at 642 nm and the emission at 661 nm was detected using a fiber fluorimeter. A linear range of 25–100, 50–200 and 25–100 μM was obtained for CP, CM and IP, respectively. The sensitivity for both CP and CM was 0.14 while it was 0.06 mV/μM for IP. The detection limit was 1.25 μM in all cases. In another approach the detection of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) was investigated based on chemiluminescence detection. The assay was based on competition between the binding of horseradish peroxidase (HRP) labeled versus unlabeled 2,4-D, on immobilized anti-2,4-D monoclonal antibodies (mAbs). Two other peroxidases from tomato (MOP) and tobacco (TOP) were also employed as labels for the 2,4-D assays. The assays were optimized by testing various chemiluminescent substrates for improving the signal intensity and sensitivity of the assays. Based on colorimetry a linear range of 0.35–20 and 0.7–4 μg/ml was obtained for TOP and MOP conjugates, respectively. However, using chemiluminometry both TOP and MOP showed a linear range of 0.5–5000 ng/ml. TOP demonstrated a higher specific activity compared to MOP and HRP. A detection limit of 50 pg/ml for 2,4-D was obtained using TOP conjugate and chemiluminescence detection. Both the fluorimetric and chemiluminometric assays were carried out in glass microcapillaries and detected using a computer coupled photomultiplier tube (PMT) as a photon detector. The responses were also compared with microtitre plate based colorimetric assay.