A fluorescent biosensor is synthesized and described. The biosensor consists of polyelectrolyte microcapsules with glucose oxidase (GOx) entrapped in the cavities and an oxygen-sensitive fluorescent indicator Ru(dpp) immobilized in shells, where Ru(dpp) is tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride. The theoretical activity of the encapsulated GOx and the effect storage time and medium composition have on the stability of sensor microcapsules are determined from polarographic measurements. No change in the activity of the encapsulated enzyme and or its loss to the storage medium are detected over the test period. The dispersion medium (water or a phosphate buffer) are shown to have no effect on the activity of microcapsules with immobilized GOx. The described optical sensor could be used as an alternative to electrochemical sensors for in vitro determination of glucose in the clinically important range of concentrations (up to 10 mmol/L).

A polyelectrolyte-based enzymatic diagnosticum with a precipitation detection system that can be used as a biosensor was created. The detection method was based on the change in polyelectrolyte microcapsule weight with respect to the urea content. The possibility of biosensor reutilization was demonstrated. The appropriate ionic precipitator causing precipitation of insoluble carbonate within the microcapsules and the optimal microcapsule titre were found. In the solution of monovalent anions (chlorides), the activity of encapsulated urease was shown to increase monotonically as the square root of the ionic strength depending on the elevation of the salt content. The activity drastically increased in a narrow concentration interval (0.6–0.8 mM) of divalent anions (sulfates) and reached the level of the native enzyme activity.

The review is focused on a new, unique and promising method of creating a precision local temperature gradient in the micro- and nanovolumes, allowing to heat a single cell and to explore exogenous and endogenous intracellular processes. Retrospective analysis and systematization of advanced developments in the study of intracellular temperature have been carried out. A device is described in detail consisting of an optical nanoheater, which uses metallic nanoparticles or water warming up with infrared laser beam, enabling setting a stationary temperature gradient of up to 70°C at a distance of 20 μm from the heat source, and a fluorescent microthermometer, which can measure temperature in microvolumes with millisecond resolution. Special attention is paid to the physical, in particular, thermodynamic description of temperature as а parameter of macro-, micro- and nanosystems and to the description of the ultralocal temperature gradient induction mechanism. The results are collected on the implementation of the local heating on living cell and on the thermoinduction of intracellular processes, among which the growth rate of neurites, of about 10 μm/min in a thermal gradient is the most impressive; absolutely new prospects opened by “thermal manipulation” of a living cell are highlighted.

Human heat shock protein Hsp70 was experimentally inserted into polyelectrolyte microcapsules. Encapsulated recombinant Hsp70 was studied in terms of its effects on neutrophil apoptosis, the production of reactive oxygen species, and the secretion of tumor necrosis factor alpha by promonocytic THP-1 cells. It was found that encapsulated Hsp70 effectively inhibits neutrophil apoptosis, unlike free exogenous protein used in solution. In THP-1 cells, encapsulated and free Hsp70 reduced LPS-induced tumor necrosis factor alpha production with a similar efficiency. Encapsulated Hsp70 reduces LPS-induced reactive oxygen species production by neutrophils in the course of its release from the microcapsules but not as much as free Hsp70. Thus, the polyelectrolyte microcapsules can be used as containers for the effective delivery of Hsp70 to neutrophils and monocytes to significantly improve the functioning of the innate immune system.

The possibility of the effective delivery of lactoferrin to the cells of the innate immune system (neutrophils and monocytes) by means of biodegradable polyelectrolyte microcapsules has been shown. A study of the effect of the structural component of microcapsules, poly-L-arginine, has shown that the production of reactive oxygen species (ROS) was maximum at a concentration of the polyelectrolyte of 0.5 μg/mL. Increasing the polycation concentration significantly decreased the neutrophil viability and ROS production. The study of neutrophil apoptosis showed that hollow microcapsules containing no lactoferrin accelerated the apoptosis of phagocytes by 15–20%. Encapsulated lactoferrin inhibited the neutrophil apoptosis by 65–70%, whereas nonencapsulated lactoferrin decreased it by 35%. It was found that encapsulated lactoferrin reduced the production of TNF-α by THP-1 promonocytic cells to an approximately the same degree as nonencapsulated lactoferrin.

The stability of polymeric products to an oxidizing and hydrolysis is one of the important factors describing their weather fastness [1]. However, recently oxidizing and hydrolytic destructions began to apply for surface modification of materials with the purpose of obtaining products with the improved operation properties [2,3]. In this case process of destruction is necessary to stop at the reliable stage so that there was no noticeable change of the complex of positive properties of a polymeric product. All this requires careful learning of kinetic and diffusive regularities having place at degradation, with the purpose of improvement of properties of a surface of a product without deterioration of their bulk properties.

Microencapsulated heat shock proteins HSP 70 were studied in terms of their effects on neutrophil apoptosis, production of reactive oxygen species, and secretion of TNF-α by human neurtrophils and monocytes. Encapsulated HSP70 inhibited neutrophil apoptosis by 65% as compared to the effect of nonencapsulated HSP70; TNF-α production by the promonocytic THP-1 cells was similarly inhibited by the non-encapsulated and encapsulated HSP70. Thus, the polyelectrolyte micromolecules can be used as containers for effective delivery of HSP70 up to neutrophils and monocytes to correct the innate immunity functions.

Enzyme based micron sized sensing system with optical readout was fabricated by co-encapsulation of urease and dextran couple with pH sensitive dye SNARF-1 into polyelectrolyte multilayer capsules. Co-precipitation of calcium carbonate, urease and dextran followed up by multilayer film coating and Ca-extracting by EDTA resulted in formation of 3.5-4 micron capsules, what enable the calibrated fluorescence response to urea in concentration range from 10(-6) to 10(-1) M. Sensitivity to urea in concentration range of 10(-5)-10(-1) M was monitored on capsule assemblies (suspension) and on single capsule measurements. Urea presence can be monitored on single capsule level as illustrated by Confocal fluorescent microscopy.

Microcapsules composed of synthetic (sodium polystyrene sulfonate and polyallylamine hydrochloride) and biodegradable polyelectrolytes (dextran sulfate and polyarginine hydrochloride) deposited on carbonate microparticles have been obtained. The ultrastructural organization of biodegradable microcapsules has been studied by transmission electron microscopy. The shell of biodegradable microcapsules is well formed even after the deposition of six polyelectrolyte layers and has an average thickness of 44 ± 3.0 nm; their inner polyelectrolyte matrix is less branched than that of synthetic microcapsules. By using spectroscopy, the efficiency of the encapsulation of FITC-labeled BSA by adsorption depending on the number of PE layers in the capsule has been estimated. It has been shown that the maximum amount of the protein is incorporated into capsules comprising six and seven polyelectrolyte layers (4 and 2 pg/capsule, respectively). It has been concluded that the adsorption of proteins into preformed polyelectrolyte capsules enables one to avoid protein losses that occur with the method in which biomineral cores obtained by coprecipitation are used for encapsulation.

The effect of encapsulated in polyelectrolyte microcapsules heat shock protein on production of reactive oxygen species (ROS) by RAW264 was investigated. It is shown that the interaction of microcapsules (with Hsp70) with RAW264 there is a decrease ROS production.

The distribution of bovine serum albumin and ferritin within polyelectrolyte microcapsules was studied by transmission electron and confocal microscopy at the pH range 2–5. It was estimated that the protein’s distribution depends on the isoelectric point (pI) and first polyelectrolyte used for the preparation of the capsule shell. The peptide is placed in the bulk of capsule if the pH values of the medium are close to the isoelectric point of the protein and polycation was used as a first polyelectrolyte layer. If the first polyelectrolyte was polyanion, the protein is located near the internal surface of the shell. The protein is situated near the internal surface of the shell for both polyelectrolytes when pH is equal to pI.

Using the methods of light scattering and optical microscopy, data have been obtained on the thermosensitivity of polyelectrolyte microcapsules formed of alternating layers of polyallylamine and polystyrenesulfonate, hollow and with included polyelectrolyte complexes and proteins. It is shown that all three types of capsule shrink with increasing temperature and time interval of thermal influence, and their diameter decreases. It is proposed that the thermosensitivity of microcapsules be estimated by the temperature factor of the rate of their shrinkage ( E s ). For all three types of microcapsule containing from 6 to 10 layers in the shell, the phenomenon of alternant thermosensitivity depending on the number of shell layers is revealed—with an odd number of layers the shrinkage is stronger than with an even one. Using the transport proteins of blood—hemoglobin and bovine serum albumin—as an example, the dependence of the thermosensitivity of microcapsules on the quantity, the degree of ionization, and the conformational state of the encapsulated protein has been investigated.

Electron micrographs of ultrathin sections of polyelectrolyte microparticles (PE microparticles) containing and not containing proteins were obtained and analyzed. CaCO3 microspherolytes were used as a core basis for these particles. The data indicate that protein-free PE microparticles are entities with a complex internal organization, containing a set of filamentary and closed nanoelements of polyelectrolyte nature. It was found that in PE microcapsules containing protein, unlike those without protein, polyelectrolytes are present only in the near-the-surface layer, and the external, spatially arranged shell encloses the internal volume filled with a protein solution. A stracture of nanolayered shells of polyelectrolyte microcapsules has been investigated with fluorescent probe merocyanine-540 (M540). A structural scheme for microcapsules' shells has been proposed. Data on thermal sensitivity of 3 types polyelectrolyte microcapsules: hollow, encapsulated with interpolyelectrolyte complexes and proteins were obtained with light scattering and optical microscopy.

A new type of a wide-purpose diagnosticum including a medical use is developed. The major element of the diagnosticum is a multilayer polyelectrolyte nanoand microcapsule with included enzyme, which can provide qualitative and quantitative information on its substrate, inhibitor or activator present in the medium to be analyzed. Compared to enzymatic methods for analysis of biological fluids, available in medicine, the microdiagnosticum we propose has many advantages. An encapsulated enzyme, first, retains its activity for at least several months, whereas the activity of a "free" enzyme in solution falls actually to zero in several days, second, retains the activity in proteinase-containing biological fluids under analysis thus eliminating the need to remove proteinases from the medium, third, it can be used repeatedly thus decreasing consumption of enzyme.