Coating and selective deposition of nanofilm on silicone rubber for cell adhesion and growth

A recently developed method for surface modification, layer-by-layer (LbL) assembly, has been applied to silicone, and its ability to encourage endothelial cell growth and control cell growth patterns has been examined. The surfaces studied consisted of a precursor, with alternating cationic polyethyleneimine (PEI) and anionic sodium polystyrene sulfonate (PSS) layers followed by alternating gelatin and poly-d-lysine (PDL) layers. Film growth increased linearly with the number of layers. Each PSS/PEI bilayer was 3 nm thick, and each gelatin/PDL bilayer was 5 nm thick. All layers were more hydrophilic than the unmodified silicone rubber surface, as determined from contact angle measurements. The contact angle was primarily dictated by the outermost layer. Of the coatings studied, gelatin was the most hydrophilic. A film of (PSS/PEI)₄/(gelatin/PDL)₄/ gelatin was highly favorable for cell adhesion and growth, in contrast to films of (PSS/PEI)₈ or (PSS/PEI)₈/PSS. Cell growth patterns were successfully controlled by selective deposition of microspheres on silicone rubber, using microcontact printing with a silicone stamp. Cell adhesion was confined to the region of microsphere deposition. These results demonstrate that the LbL self-assembly technique provides a general approach to coat and selectively deposit films with nanometer thickness on silicone rubber. Furthermore, they show that this method is a viable technique for controlling cellular adhesion and growth.     

Micropatterned polyelectrolyte nanofilms promote alignment and myogenic differentiation of C2C12 cells in standard growth media

Alignment of skeletal myoblasts is considered a critical step during myotube formation. The C2C12 cell line is frequently used as a model of skeletal muscle differentiation that can be induced by lowering the serum concentration in standard culture flasks. In order to mimic the striated architectures of skeletal muscles in vitro, micro‐patterning techniques and surface engineering have been proven as useful approaches for promoting elongation and alignment of C2C12 myoblasts, thereby enhancing the outgrowth of multi‐nucleated myotubes upon switching from growth media (GM) to differentiative media (DM). Herein, a layer‐by‐layer (LbL) polyelectrolyte multilayer deposition was combined with a micro‐molding in capillaries (MIMIC) method to simultaneously provide biochemical and geometrical instructive cues that induced the formation of tightly apposed and parallel arrays of differentiating myotubes from C2C12 cells maintained in GM media for 15 days. This study focuses on two different types of patterned/self‐assembled nanofilms based on alternated layers of poly (allylamine hydrochloride) (PAH)/poly(sodium 4‐styrene‐sulfonate) (PSS) as biocompatible but not biodegradable polymeric structures, or poly‐L ‐arginine sulfate salt (pARG)/dextran sulfate sodium salt (DXS) as both biocompatible and biodegradable surfaces. The influence of these microstructures as well as of the nanofilm composition on C2C12 skeletal muscle cells' differentiation and viability was evaluated and quantified, pointing to give a reference for skeletal muscle regenerative potential in culture conditions that do not promote it. At this regard, our results validate PEM microstructured devices, to a greater extent for (PAH/PSS)₅‐coated microgrooves, as biocompatible and innovative tools for tissue engineering applications and molecular dissection of events controlling C2C12 skeletal muscle regeneration without switching to their optimal differentiative culture media in vitro. Biotechnol. Bioeng. 2013; 110: 586–596. © 2012 Wiley Periodicals, Inc.     

Spray Deposition of Silver Nanowire Electrodes for Semitransparent Solid‐State Dye‐Sensitized Solar Cells

Transparent top electrodes for solid‐state dye‐sensitized solar cells (ssDSCs) allow for fabrication of mechanically stacked ssDSC tandems, partially transparent ssDSCs for building integration, and ssDSCs on metal foil substrates. A solution‐processed, highly transparent, conductive electrode based on PEDOT:PSS [poly(3,4‐ethylenedioxythiophene):poly(styrenesulfonate)] and spray‐deposited silver nanowires (Ag NWs) is developed as an effective top contact for ssDSCs. The electrode is solution‐deposited using conditions and solvents that do not damage or dissolve the underlying ssDSC and achieves high performance: a peak transmittance of nearly 93% at a sheet resistance of 18 Ω/square – all without any annealing that would harm the ssDSC. The role of the PEDOT:PSS in the electrode is twofold: it ensures ohmic contact between the ssDSC 2,2′,7,7′‐tetrakis‐(N,N‐di‐p‐methoxyphenylamine)9,9′‐spirobifluorene (Spiro‐OMeTAD) overlayer and the silver nanowires and it decreases the series resistance of the device. Semitransparent ssDSCs with D35 dye fabricated using this Ag NW/PEDOT:PSS transparent electrode show power conversion efficiencies of 3.6%, nearly as high as a reference device using an evaporated silver electrode (3.7%). In addition, the semitransparent ssDSC shows high transmission between 700–1100 nm, a necessity for use in efficient tandem devices. Such an electrode, in combination with efficient ssDSCs or hybrid perovskite‐sensitized solar cells, can allow for the fabrication of efficient, cost‐effective tandem photovoltaics.     

Immobilization of lutetium bisphthalocyanine in nanostructured biomimetic sensors using the LbL technique for phenol detection

This study describes the development of amperometric sensors based on poly(allylamine hydrochloride) (PAH) and lutetium bisphthalocyanine (LuPc(2)) films assembled using the Layer-by-Layer (LbL) technique. The films have been used as modified electrodes for catechol quantification. Electrochemical measurements have been employed to investigate the catalytic properties of the LuPc(2) immobilized in the LbL films. By chronoamperometry, the sensors present excellent sensitivity (20 nA μM(-1)) in a wide linear range (R(2)=0.994) up to 900 μM and limit of detection (s/n=3) of 37.5 × 10(-8)M for catechol. The sensors have good reproducibility and can be used at least for ten times. The work potential is +0.3 V vs. saturated calomel electrode (SCE). In voltammetry measurements, the calibration curve shows a good linearity (R(2)=0.992) in the range of catechol up to 500 μM with a sensitivity of 90 nA μM(-1) and LD of 8 μM.     

Quantification of arabinogalactan-protein in plant extracts by single radial gel diffusion

The amount of arabinogalactan-protein in whole plant extracts can be quantified by single radial diffusion in agarose gels containing a dye known as the beta-glucosyl-Yariv reagent which specifically interacts with and precipitates arabinogalactan-proteins. The lower limit of quantification is 0.04 microgram of arabinogalactan-protein; gum arabic is used as a standard reference arabinogalactan-protein. In principle, this method can be adapted to measure levels of any dye-precipitating macromolecule; for example, acidic polysaccharides can be estimated by their binding to the cationic dye Alcian blue.     

Fabrication of protein nanotubes based on layer-by-layer assembly

Nanotubes of cytochrome C (cyto-c) with glutaraldehyde (GA) or PSS based on the layer-by-layer (LbL) assembly through covalent binding and electrostatic adsorption have been fabricated. The combination of the template method and the LbL method for fabrication of nanotubes exhibits low cost, simplicity, and versatility. The tubular morphology of the assembled glutaraldehyde and cytochrome C film was demonstrated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) measurements.The components of the tubes were determined by energy-dispersive X- ray spectra (EDAX). It is found that the assembled tubes keep the proteins' biochemical activity and electronic activity by cyclic voltammograms. The measurements of ultraviolet spectra and circular dichroism (CD) on the assembled nanotubes confirmed the cyto-c existence in the tubes.     

Quantitative PCR in the diagnosis of Leishmania

Polymerase chain reaction (PCR) is a sensitive and rapid method for the diagnosis of canine Leishmania infection and can be performed on a variety of biological samples, including peripheral blood, lymph node, bone marrow and skin. Standard PCR requires electrophoretic analysis of the amplification products and is usually not suitable for quantification of the template DNA (unless competitor-based or other methods are developed), being of reduced usefulness when accurate monitoring of target DNA is required. Quantitative real-time PCR allows the continuous monitoring of the accumulation of PCR products during the amplification reaction. This allows the identification of the cycle of near-logarithmic PCR product generation (threshold cycle) and, by inference, the relative quantification of the template DNA present at the start of the reaction. Since the amplification product are monitored in "real-time" as they form cycle-by-cycle, no post-amplification handling is required. The absolute quantification is performed according either to an internal standard co-amplified with the sample DNA, or to an external standard curve obtained by parallel amplification of serial known concentrations of a reference DNA sequence. From the quantification of the template DNA, an estimation of the relative load of parasites in the different samples can be obtained. The advantages compared to standard and semi-quantitative PCR techniques are reduction of the assay's time and contamination risks, and improved sensitivity. As for standard PCR, the minimal components of the quantitative PCR reaction mixture are the DNA target of the amplification, an oligonucleotide primer pair flanking the target sequence, a suitable DNA polymerase, deoxynucleotides, buffer and salts. Different technologies have been set up for the monitoring of amplification products, generally based on the use of fluorescent probes. For instance, SYBR Green technology is a non-specific detection system based on a fluorescent dsDNA intercalator and it is applicable to all potential targets. TaqMan technology is more specific since performs the direct assessment of the amount of amplified DNA using a fluorescent probe specific for the target sequence flanked by the primer pair. This probe is an oligonucleotide labelled with a reporter dye (fluorescent) and a quencher (which absorbs the fluorescent signal generated by the reporter). The thermic protocol of amplification allows the binding of the fluorescent probe to the target sequence before the binding of the primers and the starting of the polymerization by Taq polymerase. During polymerization, 5'-3' exonuclease activity of Taq polymerase digests the probe and in this way the reporter dye is released from the probe and a fluorescent signal is detected. The intensity of the signal accumulates at the end of each cycle and is related to the amount of the amplification product. In recent years, quantitative PCR methods based either on SYBR Green or TaqMan technology have been set up for the quantification of Leishmania in mouse liver, mouse skin and human peripheral blood, targeting either single-copy chromosomal or multi-copy minicircle sequences with high sensitivity and reproducibility. In particular, real-time PCR seems to be a reliable, rapid and noninvasive method for the diagnosis and follow up of visceral leishmaniasis in humans. At present, the application of real-time PCR for research and clinical diagnosis of Leishmania infection in dogs is still foreseable. As for standard PCR, the high sensitivity of real-time PCR could allow the use of blood sampling that is less invasive and easily performed for monitoring the status of the dogs. The development of a real-time PCR assay for Leishmania infantum infection in dogs could support the standard and optimized serological and PCR methods currenly in use for the diagnosis and follow-up of canine leishmaniasis, and perhaps prediction of recurrences associated with tissue loads of residual pathogens after treatment. At this regard, a TaqMan Real Time PCR method developed for the quantification of Leishmania infantum minicircle DNA in peripheral blood of naturally infected dogs sampled before and at different time points after the beginning of a standard antileishmanial therapy will be illustrated.     

Sequential actions of glucose oxidase and peroxidase in molecular films assembled by layer-by-layer alternate adsorption

Molecular films of protein/polyion layers were assembled by means of alternate adsorption through electrostatic interaction. Glucose oxidase (GOD) and peroxidase (POD) were assembled in combination with sodium poly(styrenesulfonate) (PSS) and poly(ethyleneimine) (PEI), respectively. Enzyme activities of those films on specific substrates (glucose and H(2)O(2)) were examined by coloring reaction of dye DA67. A multienzyme film containing GOD layer and POD layer was prepared by alternate adsorption of POD/PSS followed by PEI/GOD. Sequential redox reaction of glucose/H(2)O(2)/DA67 was demonstrated successfully with this supramolecular system. (c) 1996 John Wiley & Sons, Inc.     

A quantitative fluorescence-based microplate assay for the determination of double-stranded DNA using SYBR Green I and a standard ultraviolet transilluminator gel imaging system

Various assays are available for quantification of DNA in solution, but none has been described that is both sensitive and specific for double-stranded (ds) DNA and features practical properties such as low dye and equipment costs, speed, and highly parallel microplate formats. Here we show that quantitative and sensitive measurement of ds DNA in solution is achieved using a 96-well microplate SYBR Green I assay and a standard uv transillumination-based gel-imaging system for detection. Specific detection of ds DNA was obtained over a broad concentration range of 0.5-500 ng using a single low dye concentration of up to 1/6250. Measured SYBR Green I fluorescence was not significantly affected by pH variation (4-10), assay volume (50-250 microliter l), and time (4-15 min), and measurements were appreciably compatile with commonly encountered concentrations of contaminating salts, organics, detergents, and other substances. ds DNA yielded up to 13-fold higher fluorescence compared to single-stranded DNA or RNA, but this ratio was dependent somewhat on GC content and fragment size. Of note, linear ds DNA fluoresced significantly stronger than supercoiled plasmid DNA. Our method should be broadly applicable for sensitive, rapid, and inexpensive ds DNA quantification in the average molecular biology laboratory.     

Significantly Enhanced Thermoelectric Properties of PEDOT:PSS Films through Sequential Post‐Treatments with Common Acids and Bases

Thermoelectric (TE) materials are important for the sustainable development because they enable the direct harvesting of low‐quality heat into electricity. Among them, conducting polymers have attracted great attention arising from their advantages, such as flexibility, nontoxicity, easy availability, and intrinsically low thermal conductivity. In this work, a novel and facile method is reported to significantly enhance the TE property of poly(3,4‐ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) films through sequential post‐treatments with common acids and bases. Compared with the as‐prepared PEDOT:PSS, both the Seebeck coefficients and electrical conductivities can be remarkably enhanced after the treatments. The oxidation level, which significantly impacts the TE property of the PEDOT:PSS films, can also be well tuned by controlling the experimental conditions during the base treatment. The optimal PEDOT:PSS films can have a Seebeck coefficient of 39.2 µV K^?1 and a conductivity of 2170 S cm^?1 at room temperature, and the corresponding power factor is 334 µW (m^?1 K^?2). The enhancement in the TE properties is attributed to the synergetic effect of high charge mobility by the acid treatment and the optimal oxidation level tuned by the base treatment.     

A colorimetric assay for determination of residual detergent levels in reconstituted membrane protein preparations

Simple and robust methods for the quantification of residual detergent in purified membrane proteins are not readily available. In this article, solubilization of precipitated dye by detergent is shown to be a facile method for the quantification of residual levels of octaethylene-glycol-mono(n-dodecyl)ether in virosomal influenza vaccine. Dye solubilization starts in the critical micellar concentration range. The method is more sensitive than an existing assay and is highly accurate and precise. The method is applicable to other detergents as well. This method of residual detergent quantification is simple and straightforward and is a useful tool for quality control of subunit vaccines.     

Studies on PVP hydrogel-supported luminol chemiluminescence: 2. Luminometer calibration and potential analytical applications.

The use of poly(N-vinyl-2-pyrrolidone) (PVP) hydrogel-supported luminol chemiluminescence (CL) for the automatic determination of hydrogen peroxide and the quantification of the antiradical capacity of Trolox is described. The hydrogel containing luminol and hemin is prepared directly on a 96-well microplate and can be stored for up to 3 months without significant decrease in CL quantum yields. Furthermore, this system can also be used as a secondary light standard for the calibration of microplate luminometers.     

Photoelectrochemical, photophysical and morphological studies of electrostatic layer-by-layer thin films based on poly(p-phenylenevinylene) and single-walled carbon nanotubes

The preparation of multilayer films based on poly(p-phenylenevinylene) (PPV) and carboxylic-functionalized single-walled carbon nanotubes (SWNT-COOH) by electrostatic interaction using the layer-by-layer (LbL) deposition method is reported herein. The multilayer build-up, monitored by UV-Vis and photoluminescence (PL) spectroscopies, displayed a linear behavior with the number of PPV and SWNT-COOH layers deposited that undergo deviation and spectral changes for thicker films. Film morphology was evaluated by AFM and epifluorescence microscopies showing remarkable changes after incorporation of SWNT-COOH layers. Films without SWNT show roughness and present dispersed grains; films with SWNT-COOH layers are flatter and some carbon nanotube bundles can be visualized. The photoinduced charge transfer from the conducting polymer to SWNT-COOH was analyzed by PL quenching either by the decrease of the emission intensity or by the presence of dark domains in the epifluorescence micrographs. Photoelectrochemical characterization was performed under white light and the films containing SWNT-COOH displayed photocurrent values between 2.0 μA cm(-2) and 7.5 μA cm(-2), as the amount of these materials increases in the film. No photocurrent was observed for the film without carbon nanotubes. Photocurrent generation was enhanced and became more stable when an intermediate layer of PEDOT:PSS was interposed between the active layer and the ITO electrode, indicating an improvement in hole transfer to the contacts. Our results indicate that these multilayer films are promising candidates as active layers for organic photovoltaic cells.     

Detection of viral aerosols by use of real-time quantitative PCR

PCR quantification is regarded as one of the most promising techniques for real-time identification of bio-aerosols. We have, therefore, validated a QPCR assay for quantification of a viral aerosol sample using the double-stranded DNA-binding dye SYBR green I, an economical alternative for quantification of target microorganisms. To achieve this objective we used mycobacteriophage D29 as model organism. Phage D29 aerosol was produced in an aerosol cabinet and then collected by use of an AGI liquid sampler. A standard curve was created by use of purified genomic DNA from the phage in liquid culture of known concentration measured by titration. To prevent false-positive results caused by formation of primer–dimers, an additional data-acquisition step was added to the three-step QPCR procedure; the new technique was called four-step QPCR. The standard curve was then used to quantify the total amount of phage D29 in liquid culture and aerosol samples. For liquid culture samples there was no significant difference (P > 0.05) between results from quantification of the virus using double-agar culture and QPCR. For aerosol samples, however, the result determined by the QPCR method was significantly (P < 0.05) higher than that from the double-agar culture method. The four-step SYBR green I QPCR method is a quick quantitative method for mycobacteriophage D29 aerosol. We believe that QPCR using SYBR green I dye will be an economical method for detection of airborne bio-aerosols.     

Charge Formation in Pentacene Layers During Solar‐Cell Fabrication: Direct Observation by Electron Spin Resonance

Microscopic characterization of charge carriers in solar cells is useful for high‐performance cell fabrication because the formation and accumulation of charges in cells greatly affect the device performance. Electron spin resonance (ESR) is suitable for such characterization because it can directly observe charge carriers with spins in these cells. In this work, the ESR method is applied to organic thin‐film solar cells to investigate charge formation in such devices. Heterojunction cells of indium tin oxide (ITO)/poly(3,4‐ethylenedioxythiophene):poly(4‐styrenesulfonate) (PEDOT:PSS)/pentacene/C₆₀/bathocuproine (BCP)/Al are investigated. Clear ESR signals are observed by inserting a typical PEDOT:PSS hole buffer layer. From analysis of the dependence of the ESR characteristics on the external magnetic field direction, the bias voltage, and the duration of solar‐simulated irradiation, the charges (mobile holes) in pentacene layers are successfully identified and it can be deduced that these holes are formed at the PEDOT:PSS/pentacene interface during device fabrication. This ESR analysis provides useful knowledge for understanding device operation and improving device performance at the microscopic level.     

A Mechanistic Model of PCR for Accurate Quantification of Quantitative PCR Data

Background

Quantitative PCR (qPCR) is a workhorse laboratory technique for measuring the concentration of a target DNA sequence with high accuracy over a wide dynamic range. The gold standard method for estimating DNA concentrations via qPCR is quantification cycle () standard curve quantification, which requires the time- and labor-intensive construction of a standard curve. In theory, the shape of a qPCR data curve can be used to directly quantify DNA concentration by fitting a model to data; however, current empirical model-based quantification methods are not as reliable as standard curve quantification.

Principal Findings

We have developed a two-parameter mass action kinetic model of PCR (MAK2) that can be fitted to qPCR data in order to quantify target concentration from a single qPCR assay. To compare the accuracy of MAK2-fitting to other qPCR quantification methods, we have applied quantification methods to qPCR dilution series data generated in three independent laboratories using different target sequences. Quantification accuracy was assessed by analyzing the reliability of concentration predictions for targets at known concentrations. Our results indicate that quantification by MAK2-fitting is as reliable as standard curve quantification for a variety of DNA targets and a wide range of concentrations.

Significance

We anticipate that MAK2 quantification will have a profound effect on the way qPCR experiments are designed and analyzed. In particular, MAK2 enables accurate quantification of portable qPCR assays with limited sample throughput, where construction of a standard curve is impractical.     

Density quantification of collagen grafted on biodegradable polyester: its application to esophageal smooth muscle cell

An improved technique for quantification of collagen immobilized on polymeric substrates is needed as tissue engineering evolves. Current immobilized protein quantification methods are indirect, time-consuming, and/or inaccurate. In this study, Sirius red colorimetric microassay was shown to be feasible for quantifying the density of collagen immobilized on aminolyzed poly(L-lactic acid) (PLLA) surfaces using the specific bonding of Sirius dye to collagen. It offers a number of advantages over traditional methods, including direct staining, high sensitivity, and high stability of the dye. The detection limit is approximately 0.1 microg/cm(2), and the dynamic range is greater than 50. Sirius red dye has not been used previously for quantification of protein immobilized on polymers. The collagen densities achieved with each of the two crosslinking reagents investigated, namely glutaraldehyde (GA) and genipin, were compared. The latter is an alternative crosslinker derived from a traditional Chinese medicine. The collagen densities immobilized by the two reagents were measured to be similar. This was confirmed by the similar behaviors of esophageal primary smooth muscle cells (ESMCs) on these two modified PLLA membranes; collagen grafted with either coupler was found to greatly promote, to a similar extent, cell attachment and both short-term (4 days) and long-term (12 days) proliferation compared with unmodified PLLA. Smooth muscle cells on both modified membranes were stained to display contractile alpha-actin protein filaments.     

Real-time PCR provides improved detection and titer determination of bacteriophage

The plaque assay is the traditional method for the quantification of bacteriophage, particularly for lambda cloning vectors. Unfortunately, this technique is fraught with procedural difficulties, and the quality of the data obtained from this "gold standard" assay may be inaccurate due to the subjective interpretation of the results. The application of quantitative real-time PCR (QPCR) technology can address these issues and be a more accurate platform to evaluate phage growth conditions and quantify viral titers in phage preparations. QPCR, with an improved primer set specific for lambda phage and coupled with fluorescent dye detection of PCR products, was used to detect and quantify phages in lysates with no prior DNA purification. Phages were detected below one plaque-forming unit, and at least 89 viral copies were detected from a purified DNA sample. When unknown concentrations of various phage preparations were assessed using QPCR, they were attained more efficiently, with greater sensitivity and precision, and the method produced more accurate quantitative data spanning a wider linear range than those obtained by the plaque assay (six logs vs. one log, respectively). Finally, QPCR for the detection of phage has multiple applications, including conventional cloning and in alternative fields of study such as environmental sciences.     

Layer-by-layer self-assembly of chitosan and poly(γ-glutamic acid) into polyelectrolyte complexes

Chitosan (Ch) is a nontoxic and biocompatible polysaccharide extensively used in biomedical applications. Ch, as a polycation, can be combined with anionic polymers by layer-by-layer (LbL) self-assembly, giving rise to multilayered complexed architectures. These structures can be used in tissue engineering strategies, as drug delivery systems, or artificial matrices mimicking the extracellular microenvironment. In this work, Ch was combined with poly(γ-glutamic acid) (γ-PGA). γ-PGA is a polyanion, which was microbially produced, and is known for its low immunogenic reaction and low cytotoxicity. Multilayered ultrathin films were assembled by LbL, with a maximum of six layers. The interaction between both polymers was analyzed by: ellipsometry, quartz crystal microbalance with dissipation, Fourier transform infrared spectroscopy, atomic force microscopy, and zeta potential measurements. Ch/γ-PGA polyelectrolyte multilayers (PEMs) revealed no cytotoxicity according to ISO 10993-5. Overall, this study demonstrates that Ch can interact electrostatically with γ-PGA forming multilayered films. Furthermore, this study provides a comprehensive characterization of Ch/γ-PGA PEM structures, elucidating the contribution of each layer for the nanostructured films. These model surfaces can be useful substrates to study cell-biomaterial interactions in tissue regeneration.     

Protein-filled polyelectrolyte microcapsules in the design of enzymic microdiagnostics

Encapsulation of enzymes (lactate dehydrogenase and urease) in polyelectrolyte shells was assessed with a view to designing enzymic microdiagnostics for low-molecular compounds in native biological fluids. Polyelectrolyte microcapsules were prepared with two polyanions [poly(styrenesulfonate) PSS and dextran sulfate DS] and two polycations [poly(allylamine) PAA and poly(diallyldimethylammonium) PDADMA]; calcium carbonate microspherulites with embedded enzymes served as “cores.” It was demonstrated that the main problem in making such a biosensor is to select a pair of oppositely charged polyelectrolytes that would be optimal for enzyme functioning. The best pairs were PAA/DS and PAA/PSS for lactate dehydrogenase, and PSS/PAA and PSS/PDADMA for urease. We designed and prepared enzyme-containing microcapsules differing in polyelectrolyte composition and number of layers, and investigated their properties.