Finding the Light Emission Stimulator of Neonothopanus nambi Basidiomycete and Studying Its Properties.

A stimulator of light emission of the fungus was found in an aqueous extract from mycelium of the luminous basidiomycete Neonothopanus nambi after its treatment with ß-glucosidase. The addition of the extract to the luminous mycelium increases the level of light emission from several times to 1.5 orders of magnitude or more. The luminescence stimulator is a low-molecular-weight thermostable compound: it is detected in the permeate after filtering the extract through a 10-kDa cutoff membrane and it retains the stimulating effect after heat treatment at 100°C for 5 min. In the absorption spectrum of the aqueous sample of the stimulator, two main peaks are observed in the shortwave region (205 and 260 nm) and a shoulder in the range of 350-370 nm can be seen. The luminescence stimulator exhibits blue fluorescence with an emission maximum at 440 nm when excited at 360 nm. It was established that the luminescence-stimulating component is not a substrate (or its precursor) of the luminescent system of the N. nambi fungus.

Biodistribution of Detonation Synthesis Nanodiamonds in Mice after Intravenous Administration and Some Biochemical Changes in Blood Plasma.

Biodistribution of nanodiamonds in mice after intravenous administration, activities of AST and ALT, and the level of bilirubin in the blood plasma were studied in 2.5 h and 10, 35, and 97 days after injection of nanodiamonds. In 2.5 h after intravenous injection, nanodiamonds mainly accumulate in the lungs and liver. Then, redistribution of nanodiamonds from all organs to the liver was observed. Activities of AST and ALT and the level of bilirubin in the blood increased after 2.5 h and then decreased to the initial values.

Reusable System for Phenol Detection in an Aqueous Medium Based on Nanodiamonds and Extracellular Oxidase from Basidiomycete Neonothopanus nambi.

A reusable system for phenol determination in an aqueous medium was obtained by adsorption of extracellular oxidase from fungus Neonothopanus nambi onto modified nanodiamonds (MND) synthesized by detonation. It was found that the enzyme strongly binds to MND and exhibits catalytic activity in the reaction of co-oxidation of phenol with 4-aminoantipyrine without the addition of hydrogen peroxide. In the presence of the MND-oxidase complex, a significantly (by an order of magnitude) higher yield of the reaction product is recorded as compared to the yield in the presence of a free enzyme; the mechanism of the revealed effect is discussed. Model experiments have demonstrated the multiple use of the MND-oxidase complex for testing phenol in aqueous samples. The immobilized enzyme exhibits functional activity during long-term (2 months) storage of the MND-oxidase complex at 4°C. The data obtained create the prerequisites for using the created system in environmental monitoring of water pollution with phenol.

Extracellular Oxidases of Basidiomycete Neonothopanus nambi: Isolation and Some Properties.

Using the original technique of treating biomass with ß-glucosidase, a pool of extracellular fungal enzymes was obtained for the first time from the mycelium of basidiomycete Neonothopanus nambi. Two protein fractions containing enzymes with oxidase activity were isolated from the extract by gel-filtration chromatography and conventionally called F1 and F2. Enzyme F1 has a native molecular weight of 80-85 kDa and does not contain chromophore components; however, it catalyzes the oxidation of veratryl alcohol with Km = 0.52 mM. Probably, this enzyme is an alcohol oxidase. Enzyme F2 with a native molecular weight of approximately 60 kDa is a FAD-containing protein. It catalyzes the cooxidation of phenol with 4-aminoantipyrine without the addition of exogenous hydrogen peroxide, which distinguishes it from the known peroxidases. It was assumed that this enzyme may be a mixed-function oxidase. F2 oxidase has Km value 0.27 mM for phenol. The temperature optimums for oxidases F1 and F2 are 22-35 and 55-70°C, and pH optimums are 6 and 5, respectively.

Creation of Bifunctional Indicating Complex Based on Nanodiamonds and Extracellular Oxidases of Luminous Fungus Neonothopanus nambi.

A bifunctional indicating complex was created by immobilization of extracellular oxidases (glucose oxidase and peroxidases) of luminous fungus Neonothopanus nambi onto modified nanodiamonds (MNDs) synthesized by detonation. It was found that the enzymes firmly adsorb onto MND particles and exhibit their catalytic activity. Model in vitro experiments showed that the created MND-enzymes complex is suitable for repeated use for analyte (glucose and phenol) testing and retains its activity after storage at 4°C in deionized water for 1 month. The data obtained offer the prospects for developing a new class of reusable multifunctional indicating and diagnostic test systems on the basis of MNDs and higher fungal enzymes for medical and ecological analytics.

Detection of Hispidin by a Luminescent System from Basidiomycete Armillaria borealis.

In in vitro experiments, the possibility of using a luminescent system extracted from the luminous fungus Armillaria borealis has been shown to detect and determine the concentration of hispidin. A linear dependence of the luminescent response on the content of hispidin in solutions in the concentration range of 5.4 × 10-5-1.4 × 10-2 µM was detected. The stability of the enzyme system and the high sensitivity of the bioluminescent reaction allows carrying out multiple measurements with the analyte detection limit of 1.3 × 10-11 g. The obtained results show the prospects of creating a rapid bioluminescent method for the analysis of medical substances or extracts from various biological objects for the presence of hispidin.

Detection of nanodiamonds in biological samples by EPR spectrometry.

In model experiments in vitro, the applicability of the EPR spectrometry method for the detection of modified nanodiamonds (MNDs) in blood and homogenates of mouse organs has been established. A characteristic signal (g = 2.003, ΔH ≈ 10 G) is observed in the samples of biomaterials containing MNDs, the intensity of which increases linearly with the concentration of nanoparticles in the range of 1.6-200 µg MNDs per 1 mL of the sample. The EPR method in biomaterials reveals the presence of intrinsic paramagnetic centers, signals from which are superimposed on the signal from the MNDs. However, the intensity of these signals is small, which makes it possible to register the MNDs using EPR spectrometry with the necessary accuracy. The data obtained open up the prospects of using the EPR method for studies of the interorgan distribution, accumulation, and elimination of MNDs during their intravenous injection into experimental animals.

Adsorption of viral particles from the blood plasma of patients with viral hepatitis on nanodiamonds.

Adsorption of viral particles from the blood plasma of patients with viral hepatitis B and C on modified nanodiamonds (MNDs) was shown in the in vitro experiments. PCR method showed the treatment of plasma with MNDs leads to a decrease in the viral load by 2-3 orders of magnitude or more in both cases studied. These results make it possible to predict the applicability of MNDs for the development of new technologies of hemodialysis and plasmapheresis for binding and removal of viral particles from the blood of infected patients.

[Total Peroxidase and Catalase Activity of Luminous Basidiomycetes Armillaria borealis and Neonothopanus nambi in Comparison with the Level of Light Emission].

The peroxidase and catalase activities in the mycelium of luminous basidiomycetes Armillaria borealis and Neonothopanus nambi in normal conditions and under stress were compared. An increase in the luminescence level was observed under stress, as well as an increase in peroxidase and catalase activities. Moreover, the peroxidase activity in extracts of A. borealis mycelium was found to be almost one and a half orders of magnitude higher, and the catalase activity more than two orders of magnitude higher in comparison with the N. nambi mycelium. It can be suggested that the difference between the brightly luminescent and dimly luminescent mycelium of N. nambi is due to the content of H2O2 or other peroxide compounds.

Field emission luminescence of nanodiamonds deposited on the aligned carbon nanotube array.

Detonation nanodiamonds (NDs) were deposited on the surface of aligned carbon nanotubes (CNTs) by immersing a CNT array in an aqueous suspension of NDs in dimethylsulfoxide (DMSO). The structure and electronic state of the obtained CNT-ND hybrid material were studied using optical and electron microscopy and Infrared, Raman, X-ray photoelectron and near-edge X-ray absorption fine structure spectroscopy. A non-covalent interaction between NDs and CNT and preservation of vertical orientation of CNTs in the hybrid were revealed. We showed that current-voltage characteristics of the CNT-ND cathode are changed depending on the applied field; below ~3 V/µm they are similar to those of the initial CNT array and at the higher field they are close to the ND behavior. Involvement of the NDs in field emission process resulted in blue luminescence of the hybrid surface at an electric field higher than 3.5 V/µm. Photoluminescence measurements showed that the NDs emit blue-green light, while blue luminescence prevails in the CNT-ND hybrid. The quenching of green luminescence was attributed to a partial removal of oxygen-containing groups from the ND surface as the result of the hybrid synthesis.

Designing a reusable system based on nanodiamonds for biochemical determination of urea.

A reusable system including urease covalently bound to the surface of modified nanodiamonds (MNDs) has been developed for the multiple determination of urea. The immobilized enzyme exhibits functional activity and catalyzes the hydrolysis of urea to yield ammonia. The presence of ammonia is confirmed by the formation of a colored product after the addition of chemical reagents. It was shown that the MNDs-urease complex can function in a wide range of temperatures and pH as well as in deionized water. The complex provides a linear yield of the product at low analyte concentrations and allows the multiple determination of urea in vitro.

Effects of nanodiamonds of explosive synthesis on the skin of experimental animals locally exposed to cobalt and chrome ions.

Experiments in vivo demonstrated the protective effect of modified nanodiamonds on guinea pig skin after local exposure cobalt ions, but not chrome ions. The observed differences are determined by different adsorption of these ions by nanodiamonds: in vitro experiments showed that nanodiamonds adsorbed cobalt ions, but not chrome ions from water solutions. The perspectives of using modified nanodiamonds as a new adsorbent for prevention of allergic contact dermatitis induced by ions of bivalent metals are discussed.

Effects of modified detonation nanodiamonds on the biochemical composition of human blood.

In vitro experiments showed that protein and non-protein components of human blood serum could be absorbed on the surface of modified nanodiamonds obtained by detonation synthesis. The prospects of using nanodiamond as a new absorbent for hemodialysis, plasmapheresis, and laboratory diagnostics are discussed.

[The effect of modified nano-diamonds of detonation synthesis on the protein fractions of human blood].

It is established that the modified nano-diamonds of detonation synthesis are able to bind serum proteins of human blood. The relative selectivity is established concerning the effect of modified nano-diamonds of detonation synthesis on beta2- and gamma-globulin fractions of serum. The evidence of concentration dependence of effect of modified nano-diamonds of detonation synthesis from serum proteins is established. The study results make it possible to consider modified nano-diamonds of detonation synthesis as a potential sorbent in technologies of hemodialysis, plasmapheresis, isolation of blood proteins and as a foundation for development of new systems of laboratory diagnostic.