G. V. Presnova

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.

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.

Electrochemical Biosensors Based on Horseradish Peroxidase

The principles used for the development of electrochemical biosensors based on horseradish peroxidase are described. Peroxidase is the enzyme which catalyses the oxidation of a variety of organic molecules in the presence of hydrogen peroxide. The features of this enzyme are high catalytic activity and low specificity towards second substrate as well. Horseradish peroxidase may be used as a component of active part of biosensors for the detection of hydrogen peroxide and other compounds when peroxidase is co-immobilized together with other oxidases. Also horseradish peroxidase may be used as a component of detecting system for the biosensors based on biological recognition using specific antibodies, receptors, nucleic acids. The examples of the bio-, immuno-, DNA-sensors developed for the determination of various biologically active compounds are given.

Streptavidin conjugates with gold nanoparticles for visualization of single DNA interactions on the silicon surface

Applicability of scanning electron microscopy (SEM) for visualization of individual acts of DNA hybridization with oligonucleotide probes has been investigated using gold nanoparticles as a label. DNA or oligonucleotides were labeled with biotin molecules, which were then detected in DNA duplexes using a streptavidin conjugate with gold nanoparticles. Effective imaging of DNA duplexes was possible using the conjugate prepared by covalent binding. The detection limit of the model oligonucleotide of 19 bases was 20 pg.

Oriented immobilization of antibodies and their fragments on modified silicon for the production of nanosensors

Different methods for the covalent immobilization of specific antibodies and their fragments on a silicon surface with the subsequent formation of immune complexes that consist of an immobilized monoclonal antibody, an antigen molecule, and a molecule of a second monoclonal antibody labeled with gold nanoparticles have been studied. Prostate-specific antigen (PSA), which is a molecular biomarker for prostate cancer, was used as an antigen. A covalent conjugate of the fragments of PSA-specific antibodies with gold nanoparticles has been obtained using the thiol groups of the antibodies. Scanning electron microscopy (SEM) was used for the registration of immune complexes on the surface. The high resolution of the method made it possible to detect individual immune complexes by the presence of gold nanoparticles and to calculate their number. A new method for the chemical modification of silicon by 3-aminopropyltrimetoxysilane (APTMS) and a bifunctional reagent 1,4-phenylene diisothiocyanate (PDITC) has been developed. This method provides a uniform distribution of antigen-binding centers and their availability for the formation of immune complexes. The developed immobilization method is promising for the formation of a biospecific biosensor layer based on silicon nanowires.

Screening of bacterial genes responsible for resistance to beta-lactam antibiotics using microarrays with enzymatic detection

The method of hybridization analysis on microarrays with enzymatic detection based on horseradish peroxidase is applied to screen infectious agents of nosocomial and community-acquired infections for beta-lactamase genes causing resistance to beta-lactam antibiotics. The advantages of using this method for the rapid identification of genes are demonstrated. Similarities and differences in the distribution of beta-lactamase genes identified in the infectious agents of nosocomial and community-acquired infections are revealed. The most common type of extended-spectrum beta-lactamases (ESBLs) is CTX-M. The high prevalence of extended-spectrum beta-lactamases, particularly of the TEM-1 beta-lactamase, is demonstrated. Individually or in combination with genes of TEM-1 and SVH-1 beta-lactamases, the genes of subgroup CTX-M-1 beta-lactamases were the most frequently identified in community-acquired infectious agents. There were no cases of the simultaneous detection of multiple ESBLs in community-acquired infectious agents. Much more varied combinations of beta-lactamases were identified in nosocomial infectious agents: a combination of extended-spectrum beta-lactamases and broad-spectrum beta-lactamases was identified in 62% of strains and the simultaneous presence of two different types of ESBLs was identified in 18% of strains.

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.

Conjugates of streptavidin with gold nanoparticles for the visualization of dna single interactions on the silicon surface

The potential of the method of scanning electron microscopy (SEM) to visualize the results of individual acts of DNA and oligonucleotides hybridization using gold nanoparticles as label was investigated. Molecule of biotin was introduced into DNA or oligonucleotide, and then it was detected in DNA duplex using a conjugate of streptavidin with gold nanoparticles. Effective imaging of DNA duplexes was possible using a conjugate prepared by covalent binding.. The detection limit of the model oligonucleotide of 19 bases was 20 pg.

Comparative immobilization of antibodies on modified screen-printed graphite electrodes

Immobilization of polyclonal antibodies was studied on native screen-printed graphite electrodes (SPEs) and variously modified electrodes. SPEs coated with didodecylammonium bromide (DDAB, a synthetic membranelike substance) films with gold nanoparticles gave the maximum electrochemical response. DDAB and gold nanoparticle films strongly changed the surface morphology, and the electrochemical signal became more intense and stable. This immobilization method increased the concentration of immobilized antibodies while their activity was retained. The detection limit of the enzymatic label (horseradish peroxidase) was 0.02 ng/L of sample.