A.M. Egorov

Mediatorless biosensor for H2O2 based on recombinant forms of horseradish peroxidase directly adsorbed on polycrystalline gold

Four forms of horseradish peroxidase (HRP) have been used to prepare peroxidase-modified gold electrodes for mediatorless detection of peroxide: native HRP, wild type recombinant HRP, and two recombinant forms containing six-His tag at the C-terminus and at the N-terminus, respectively. The adsorption of the enzyme molecules on gold was studied by direct mass measurements with electrochemical quartz crystal microbalance. All the forms of HRP formed a monolayer coverage of the enzyme on the gold surface. However, only gold electrodes with adsorbed recombinant HRP forms exhibited high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct electron transfer between gold and HRP. The sensitivity of the gold electrodes modified with recombinant HRPs was in the range of 1.4-1.5 A M(-1) cm(-2) at -50 mV versus Agmid R:AgCl. The response to H(2)O(2) in the concentration range 0.1-40 microM was not dependent on the presence of a mediator (i.e. catechol) giving strong evidence that the electrode currents are diffusion limited. Lower detection limit for H(2)O(2) detection was 10 nM at the electrodes modified with recombinant HRPs.

A novel, efficient regenerating method of NADPH using a new formate dehydrogenase

Abstract A NADPH regenerating system using a new, protein engineered formate dehydrogenase (FDH) is investigated. The new enzyme is the first known NAD(P)H dependent FDH. It can be successfully employed in synthesis with other enzymes requiring a NADPH regeneration. All advantages of the known NAD(H) dependent FDHs in these enzyme coupled synthesis can now be transferred to NADP(H) dependent systems.

Chemiluminescent biosensors based on porous supports with immobilized peroxidase

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 of 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 microgram/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.

Competitive ELISA of Chloramphenicol: Influence of Immunoreagent Structure and Application of the Method for the Inspection of Food of Animal Origin

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.

Direct heterogeneous electron transfer of recombinant horseradish peroxidases on gold

Clean polycrystalline gold electrodes were modified with native glycosylated horseradish peroxidases (HRP) or two different recombinant (carbohydrate free) HRPs; recombinant wild-type HRP (rec-HRP) and recombinant HRP containing a six histidine-tag at the C-terminus of the polypeptide chain (rec-HRP-His), respectively. Only the electrodes modified with the recombinant HRPs exhibited high current responses to H2O2 due to relatively rapid direct electron transfer (ET) between recombinant HRP and gold. The absence of a carbohydrate shell on rec-HRP and the additionally existing histidine-tag on rec-HRP-His improved the electrode sensitivity to H2O2 by more than 100 times if compared with the response observed at gold modified with native HRP. Rotating disk electrode experiments indicated that the heterogeneous electron transfer rates are equal to 4.7 and 7.5 s-1 for direct electron transfer between the gold electrode and rec-HRP or rec-HRP-His, respectively.

A comparative study of peroxidases from horse radish and Arthromyces ramosus as labels in luminol-mediated chemiluminescent assays

The properties of a peroxidase from Arthromyces ramosus (ARP) in the chemiluminescent reaction of luminol oxidation have been studied. These were compared with the properties of horse radish peroxidase (HRP) in the cooxidation of luminol and p-iodophenol, the enhanced chemiluminescence (ECL) reaction. By means of the stop-flow technique, ARP was shown to have an enzymatic activity toward luminol higher than that toward HRP. ARP can efficiently catalyze luminol oxidation in the absence of substrate enhancer. pH and substrate concentrations were optimized to determine ARP with the highest sensitivity. The detection limit of ARP was 5 x 10(-13) M, the same as that for HRP in the ECL reaction. The data on the use of ARP as a label in enzyme immunoassay of human IgG are presented. ARP was shown to have all the advantages of HRP as a label in chemiluminescent enzyme immunoassays: (i) high signal intensity, (ii) slow decay of luminescence, (iii) high signal/noise ratio, and (iv) as a consequence of (i)-(iii), high detection sensitivity. However, the low thermostability of ARP can limit the potential fields of its application.

Direct electron transfer in the system gold electrode–recombinant horseradish peroxidases

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, CHisrec-HRP and NHisrec-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 H2O2 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 CHisrec-HRP and NHisrec-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.

FLOW-INJECTION ENZYME-IMMUNOASSAY OF HAPTENS WITH ENHANCED CHEMILUMINESCENCE DETECTION

Abstract A non-equilibrium flow-injection enzyme immunoassay for thyroxine (model hapten) involving affinity separation of the immunocomplex of horseradish peroxidase-labelled antibodies and antigen from the free labelled antibodies is described. An enhanced chemiluminescence reaction was used for detection of horseradish peroxidase as an antibody marker. The method allows the determination of thyroxine concentrations as low as 10−11 M within 5 min and the precision is 10% (relative standard deviation).

Chemiluminescent multiassay of pesticides with horseradish peroxidase as a label

Competitive chemiluminescent immunoassay based on a combination of five antibodies was used in a combination with neural network to identify and estimate amounts of three cross-reacting s-triazines (atrazine, terbythylazine and ametryn). Antibodies with different cross-reactivity towards s-triazines were immobilized in separate wells an eight-well microtiter strip. Training of neural networks was carried out with four different learning procedures. The best topology for the data measured was a net with two hidden layers with ten neurons in the first and 15 in the second layer trained with the Schmidhuber method. s-Triazine classification of environmental samples containing various analyte mixtures was correct in 70-100% of all cases depending on the type of analyte. The test developed can be proposed as an alternative field test for multianalyte environmental monitoring.

Analysis of the binding of bispecific monoclonal antibodies with immobilized antigens (human IgG and horseradish peroxidase) using a resonant mirror biosensor

The interaction between two monoclonal antibodies (mAbs) and their corresponding bispecific antibody (bAb) with immobilized antigens has been examined using a resonant mirror biosensor (IAsys). BAbs were produced by cell fusion. The analysed panel of affinity-purified antibodies included two parental mAbs, one specific to human IgG (hIgG), and another specific to horseradish peroxidase (HRP), and a bAb derived thereof (anti-hIgG/HRP). The real-time analysis showed the drastic differences in the avidity of bivalent anti-HRP mAbs and anti-HRP shoulder of bAbs. Thus, the observed equilibrium association constant (K(ass)) of anti-HRP mAbs was about 50 times higher that of anti-HRP shoulder of bAbs. The ratio of association rate constants (k(ass)) of mAbs and bAbs was about two, due to the statistical factor of two binding sites per bivalent antibody molecule. However, the dissociation rate constant (k(diss)) of anti-HRP shoulder of bAbs was 21 times higher k(diss) of anti-HRP mAbs. The comparison with the theoretical model shows that these observations are consistent only with a situation in which bivalent binding of mAbs with immobilized HRP predominates over monovalent binding. On the contrary, the second parental mAb (anti-hIgG) did not show the increase in avidity due to bivalent binding, compared to the anti-hIgG shoulder of bAbs, suggesting that this mAb was bound monovalently to immobilized hIgG. The K(ass) values determined by solid-phase radioimmunoassay (RIA) yielded figures almost overlapping with those obtained by IAsys. The results of the comparison of bAbs and mAbs are discussed from the viewpoint of the use of bAbs in heterogeneous systems. On the other hand, these data demonstrate that real-time analysis of antibody binding parameters in IAsys biosensor is valuable for the selection of mAbs and bAbs with desired features, for different fields of application.