M.Y. Rubtsova

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.

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.

Biosensor based on a silicon nanowire field-effect transistor functionalized by gold nanoparticles for the highly sensitive determination of prostate specific antigen

We have demonstrated label-free and real-time detection of prostate specific antigen (PSA) in human serum using silicon nanowire field effect transistors (NW FETs) with Schottky contacts (Si-Ti). The NW FETs were fabricated from SOI material using high-resolution e-beam lithography, thin film vacuum deposition and reactive-ion etching processes eliminating complicated processes of doping and thermal annealing. This allowed substantial simplifying the transistors manufacturing. A new method for covalent immobilization of half-fragments of antibodies on silicon modified by 3-glycidopropyltrimethoxysilane with thiol groups and 5nm gold nanoparticles (GNPs) was established. NW FETs functionalized by GNPs revealed extremely high pH sensitivity of 70mV/pH and enhanced electrical performance in the detection of antigen due to enhanced surface/volume ratio, favorable orientation of antibody active sites and approaching the source of the electric field close to the transistor surface. Si NWFETs were applied for quantitative detection of PSA in a buffer and human serum diluted 1/100. Response time was about 5-10s, and analysis time per sample was 1min. The limit of PSA detection was of 23fg/mL, concentration range of 23fg/mL-500ng/mL (7 orders of magnitude). The PSA concentrations determined by the NW FETs in serum were compared with well-established ELISA method. The results matched well with the correlation coefficient of 0.97.

Luminol Oxidation by Hydrogen Peroxide Catalyzed by Tobacco Anionic Peroxidase: Steady‐State Luminescent and Transient Kinetic Studies

The properties of a newly isolated anionic tobacco peroxidase from transgenic tobacco plants overexpressing the enzyme have been studied with respect to the chemiluminescent reaction of luminol oxidation. These were compared to the properties of horseradish peroxidase in the cooxidation of luminol and p‐iodophenol, the enhanced chemiluminescence reaction. The pH, luminol and hydrogen peroxide concentrations were optimized for maximum sensitivity using the tobacco enzyme. The detection limit for the latter under the optimal conditions (2.5 mM luminol, 2 mM hydrogen peroxide, 100 mM Naborate buffer, pH 9.3) was about 0.1 pM, which is at least five times lower than that for horseradish peroxidase in enhanced chemiluminescence with p‐iodophenol. The rate constants for the elementary steps of the enzyme‐catalyzed reaction have been determined: k1= 4.9 × 106M−1 s1, k2= 7.3 × 106M−1 s−1, k3= 2.1 × 106M−1 s−1 (pH 9.3). The similarity of these rate constants is unusual for plant peroxidases. The high catalytic activity of tobacco peroxidase in the luminescent reaction is explained by the high reactivity of its Compound II toward luminol and the high stability of the holoenzyme with respect to heme dissociation. This seems to be a unique property of this particular enzyme among other plant peroxidases.

AN ASSAY FOR HUMAN MYOGLOBIN USING THE TECHNIQUE OF MEMBRANE IMMUNOASSAY WITH LIGHT-SENSITIVE NYLON SUPPORTS

Abstract A method for rapid determination of human myoglobin was developed. The method is based on the technique of membrane immunoassay with light-sensitive nylon membranes for covalent immobilization of antibodies. The kinetic of binding of myoglobin with immobilized antibodies was studied. The pumping of reagents through the pores of a membrane allows to reduce the total assay time to 40 min. The sensitivity of membrane assay of myoglobin was 20 ng/ml. A semi-quantitative method with visual detection also was proposed. It allows to distinguish various states of myocardial infarction pathology.

Synthesis, SAR and molecular docking study of novel non-β-lactam inhibitors of TEM type β-lactamase

The novel classes of acylated phenoxyanilide and thiourea compounds were investigated for their ability to inhibit TEM type β-lactamase enzyme. Two compounds 4g and 5c reveal the inhibition potency in micromolar range and show their action by non-covalent binding in the vicinity of the TEM-171 active site. The structure activity relationship around carbon chain length and different substituents in ortho- and para-positions of acylated phenoxyanilide as well as molecular modelling study has been performed.

Prevalence of Escherichia coli Resistant to Beta-Lactam Antibiotics among Patients with Chronic Obstructive Pulmonary Disease and Urinary Tract Infection

Urinary tract infection (UTI), which is typically caused by Escherichia coli (E. coli), is an insufficiently recognized co-morbidity among patients with chronic obstructive pulmonary disease (COPD). Adequate treatment can be complicated by resistance of the causative pathogen to beta-lactam antibiotics, which often produce beta-lactamase enzymes that destroy the antibiotic. The beta-lactamase family of enzymes is extremely diverse, including different types of enzyme and mutant forms. In this study, we analyzed 580 patients with COPD (236 females and 344 males) and thus found 218 patients with co-morbid UTIs, including 58 patients with UTI caused by E. coli (38 females and 20 males). We also investigated cases of uncomplicated symptomatic and asymptomatic UTI caused by E. coli and the presence of resistance to beta-lactam antibiotics among those patients. The E. coli strains resistant to beta-lactam antibiotics were selected for their ability to grow on selective media, before DNA microarrays were applied for specific identification of three beta-lactamase gene types (i.e., TEM, SHV and CTX-M). Overall, 83% of E. coli strains responsible for UTIs in COPD patients carried extended-spectrum beta-lactamase genes. The most prevalent were those producing CTX-M, with CTX-M-15 being predominant. The rare CTX-M-27 and TEM-15 genes were also detected in two samples. Three samples contained several extended-spectrum beta-lactamase genes simultaneously (CTX-M-15 or CTX-M-14 plus SHV-5 or TEM-15). This high prevalence of resistant E. coli strains necessitates rational antibiotic selection when treating UTI to prevent COPD exacerbations. Additionally, antibiotic therapy should be aligned with and adapted to existing and potential COPD co-morbidities.

Mutual influence of secondary and key drug‐resistance mutations on catalytic properties and thermal stability of TEM‐type β‐lactamases

Highly mutable β‐lactamases are responsible for the ability of Gram‐negative bacteria to resist β‐lactam antibiotics. Using site‐directed mutagenesis technique, we have produced in vitro a number of recombinant analogs of naturally occurring TEM‐type β‐lactamases, bearing the secondary substitution Q39K and key mutations related to the extended‐spectrum (E104K, R164S) and inhibitor‐resistant (M69V) β‐lactamases. The mutation Q39K alone was found to be neutral and hardly affected the catalytic properties of β‐lactamases. However, in combination with the key mutations, this substitution resulted in decreased KM values towards hydrolysis of a chromogenic substrate, CENTA. The ability of enzymes to restore catalytic activity after exposure to elevated temperature has been examined. All double and triple mutants of β‐lactamase TEM‐1 bearing the Q39K substitution showed lower thermal stability compared with the enzyme with Q39 intact. A sharp decrease in the stability was observed when Q39K was combined with E104K and M69V. The key R164S substitution demonstrated unusual ability to resist thermal inactivation. Computer analysis of the structure and molecular dynamics of β‐lactamase TEM‐1 revealed a network of hydrogen bonds from the residues Q39 and K32, related to the N‐terminal α‐helix, towards the residues R244 and G236, located in the vicinity of the enzymes catalytic site. Replacement of Q39 by lysine in combination with the key drug resistance mutations may be responsible for loss of protein thermal stability and elevated mobility of its secondary structure elements. This effect on the activity of β‐lactamases can be used as a new potential target for inhibiting the enzyme.

A Novel Streptavidin–luciferase Fusion Protein: Preparation, Properties and Application in Hybridization Analysis of DNA

A streptavidin–luciferase fusion protein comprising the thermostable mutant form of firefly luciferase Luciola mingrelica and minimal core streptavidin was constructed. The streptavidin–luciferase fusion was mainly produced in a tetrameric form with high luciferase and biotin‐binding activities. It was shown that fusion has the same Km values for ATP and luciferin and the bioluminescence spectra as initial luciferase. The linear dependence of the bioluminescence signal on the content of the fusion was observed within the range of 10−18–10−13 mol per well. Successful application of obtained fusion in a biospecific bioluminescence assay based on biotin–streptavidin interactions was demonstrated by the example of a specific DNA hybridization analysis. A DNA hybridization analysis for Escherichia coli cells identification was developed using unique for these cells gadB fragment encoding glutamate decarboxylase. The amplified biotinylated GadB fragments were hybridized with the immobilized oligonucleotide probes; then, the biotin in the DNA duplexes was detected using the streptavidin–luciferase fusion protein. To reach the high sensitivity of the assay, we optimized the conditions of the assay. It was shown that the use of Pluronic for plate modification resulted in a significant reduction in the DNA detection limit which finally was 0.4 ng per well.

Implementation of scanning probe microscopy for the solution of molecular diagnostics tasks

We present new approaches to improve the efficiency of DNA by scanning probe microscopy using a highly specific hybridization on affine surfaces and nanostructures of gold as a labels. Scanning probe microscopy allows to register of individual acts of hybridization by the detection of gold labels on the surface affinity followed by automatic calculation of the total.