The kinetics of the bioelectrocatalytic reduction of hydrogen peroxide has been studied at gold electrodes modified with different forms of horseradish peroxidase (HRP). Native HRP, wild type ...recombinant HRP (rec-HRP) and its two mutant forms containing a six-histidine tag at the C- or N-terminus, C
Hisrec-HRP and N
Hisrec-HRP, respectively, have been used for an adsorptive modification of the gold electrodes. The histidine sequences, i.e. histidine tags, were introduced into the peroxidase structure by genetic engineering of non-glycosylated rec-HRP using an
Escherichia coli expression system. Experiments with a gold rotating disc electrode demonstrated that electrodes with the adsorbed rec-HRP forms exhibited high and stable current response to H
2O
2 due to its bioelectrocatalytic reduction based on direct (mediatorless) ET between gold and the active site of HRP. The heterogeneous ET rate constants were evaluated to be in the order of 20 or 33 s
−1 between rec-HRP or its histidine mutants and gold, respectively, in 0.01 M phosphate buffer (pH 7.4) containing 0.15 M NaCl. The increase in the heterogeneous ET rate found for C
Hisrec-HRP and N
Hisrec-HRP is probably due to the interaction of the histidine tag with the electrode surface. The kinetic data demonstrate that new possibilities for enhancing the catalytic activity of the enzyme at the electrode
∣
solution interface can be achieved by genetic engineering design of the enzyme molecules.
Combining a sensitive and fast chemiluminescence detection method with novel immobilization methods for horseradish peroxidase (HRP) has enabled us to develop sensitive sensors for hydrogen peroxide ...and the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). HRP biospecific immobilization was shown to have well-defined advantages in terms of a higher specific activity of the enzyme on the surface and a greater sensitivity in the enhanced chemiluminescence (ECL) reaction. The lower detection limit for hydrogen peroxide was 0·025 nmol with HRP immobilized on nitrocellulose membrane through avidin-biotin linkage. An immunosensor for the detection of 2,4-D provides a lower detection limit of 0·2 μg/l with specific antibodies covalently immobilized on photoactivated nylon. The assay with chemiluminescent detection was also characterized with a wider concentration range when compared with the colorimetric assay.
An indirect competitive ELISA for the detection of chloramphenicol (CAP) in food of animal origin (milk, meat, eggs) is described. Influence of immunoreagent structure and composition on the assay ...sensitivity and specificity was investigated. Two CAP derivatives were used for conjugation with proteins: CAP succinate and a diazo derivative of CAP. Molar incorporation of CAP into the coating conjugates was also varied. To eliminate matrix effect on the assay results, a special casein-containing buffer was used for milk samples, whereas for meat and egg samples a 50-fold dilution of the buffer extracts was needed. The method developed permits CAP concentrations to be determined in the range 0.08100 μg 1
−1
. The detection limit is 0.08 μg kg
−1
. Recovery in different food samples averages between 70 and 130%. The method can be applied for inspection of food of animal origin for CAP residues.
Polyclonal antibodies against conjugates of chloramphenicol succinate and chloramphenicol base with proteins were obtained and characterized in direct ELISA. Antiserum against a conjugate of ...chloramphenicol (CAP) base with BSA (direct coupling) was very specific and showed cross-reactivity only with CAP succinate (11.3%) and CAP base (4.6%); whereas, antisera against a conjugate of CAP succinate with a protein recognized CAP succinate strongly as an initial compound. In direct ELISA, antisera against a conjugate of CAP succinate with KLH (homologous assay) and CAP base with BSA (heterologous assay) showed similar sensitivity: IC
50
were 1.3 and 1.5 ng mL
−1
, respectively. Applicability of the immunoreagents obtained was shown in the analysis of CAP residues in milk (3.5% fat content). Detection limit of 0.3 ng mL
−1
was obtained for milk diluted 5 times.
Many phenolic compounds are known to enhance the chemiluminescence associated with the horseradish peroxidase-catalyzed oxidation of luminol, but the mechanism of enhancement is still unproved. Using ...stopped-flow spectrophotometry, we have found that a series of luminescence enhancers react rapidly with the peroxidase reactive intermediates (compound I and compound II) supporting the hypothesis that the enhancement is due to the acceleration of the enzyme turnover. In addition, pulse radiolysis experiments have shown that the enhancers' phenoxyl radicals oxidize luminol, consistent with a redox mediator role for the enhancers. The latter reaction was found to be reversible, showing that enhancers of low reduction potential, which are efficient in accelerating the enzyme turnover, are also scavengers of luminol radicals and therefore luminescence quenchers. Using these data, a simple model is proposed which correctly predicts that the efficiency of a phenolic compound as luminescence enhancer depends on the reduction potential of the respective phenoxyl radical according to a bell-shaped function with a maximum at ∼0.8 V.
The influence of immunoreagents' structure on assay performance was investigated and a range of ELISAs for streptomycin in direct and indirect format was developed. Streptomycin was conjugated with ...proteins (bovine serum albumin (BSA) for immunization and ovalbumin (OVA) for immobilization on a plate) by two different methods. Streptomycin was derivatized with carboxymethoxylamine (CMO) and then coupled to a protein or the protein was activated with adipic acid dihydrazide (ADH) and then coupled with streptomycin. A conjugate with horse-radish peroxidase was synthesized using streptomycin-ADH derivative. With the indirect ELISA the most sensitive assay for polyclonal antisera against streptomycin-oxime-BSA in combination with homologous and heterologous conjugates (limit of detection 2.5 ng ml-1) was developed, whereas for a combination 'antisera against streptomycin-ADH-BSA/heterologous conjugate' higher background level of a calibration curve was observed. Besides the level was very high (about 60%) for homologous conjugate. In a direct ELISA similar sensitivity was achieved only for antisera against streptomycin-oxime-BSA (limit of detection 3.0 ng ml-1). Chemiluminescent detection allowed to increase the assay sensitivity by several times (limit of detection 0.5 ng ml-1) but led to the worse reproducibility (CV 16%). A sensitive and simple direct ELISA for analysis of streptomycin in milk products without preliminary sample preparation was developed (limit of detection 3.2 ng ml-1). In the indirect ELISA an influence of fat content of a milk product on assay performance was observed.
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