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Surface modification with polyallylamines for adhesion of biopolymers and cells
... The surface plasmon-polaritons are an example of the SWs at the metaldielectric interface [6]. Investigation of the optical SWs is an essential area of plasmonics since they play an important role in the development of chemical sensors [7,8] and label-free biosensors [9,10]. Since the optical SWs are localized at the phase boundary, they have shallow penetration depth into the bulk medium. ...
... The recombinant E. coli cells transformed with plasmid pET21-GFP were grown in the Luria-Bertani (LB) broth with 8 mg/ml chloramphenicol (Panreac, Spain) and 150 µg/mL ampicillin (Amresco, USA) until the mid-log phase (optical density at 600 nm 0.5 optical units (o.u.) per ml and then induced with 1 mM IPTG. Induced E. coli cells were immobilized onto the pre-cleaned 1D PC surface with 0.1 mg/ml pAA 65 kDa (Sigma-Aldrich, USA) [10] for 10 min at room temperature, washed with phosphate buffer solution (PBS). ...
We present a depth-localized illumination technique for wide-field fluorescence microscopy, based on long-range optical surface waves. This technique allows one to excite the fluorescence only in a thin near-substrate layer of the specimen. Our experimental setup is compatible with both upright and inverted microscopes. It provides fluorescent microscopic images, which are superior to the epifluorescence ones in signal-to-noise ratio, contrast, and detail. We demonstrate the applicability of our technique for imaging both bacterial and eukaryotic cells (E. coli and HeLa, respectively).
... The scan rate was 1 Hz. Height histograms allowed us to measure coating thickness [32]. As seen in the histogram in Figure 9a, the mean thickness of the APTES layer was 3.5 nm, and the mean height of roughness was 0.5 nm. ...
We propose and demonstrate dendrimer-based coatings for a sensitive biochip surface that enhance the high-performance sorption of small molecules (i.e., biomolecules with low molecular weights) and the sensitivity of a label-free, real-time photonic crystal surface mode (PC SM) biosensor. Biomolecule sorption is detected by measuring changes in the parameters of optical modes on the surface of a photonic crystal (PC). We describe the step-by-step biochip fabrication process. Using oligonucleotides as small molecules and PC SM visualization in a microfluidic mode, we show that the PAMAM (poly-amidoamine)-modified chip’s sorption efficiency is almost 14 times higher than that of the planar aminosilane layer and 5 times higher than the 3D epoxy-dextran matrix. The results obtained demonstrate a promising direction for further development of the dendrimer-based PC SM sensor method as an advanced label-free microfluidic tool for detecting biomolecule interactions. Current label-free methods for small biomolecule detection, such as surface plasmon resonance (SPR), have a detection limit down to pM. In this work, we achieved for a PC SM biosensor a Limit of Quantitation of up to 70 fM, which is comparable with the best label-using methods without their inherent disadvantages, such as changes in molecular activity caused by labeling.
... To bind BSA with the PC chip surface, a 0.1 mg/mL solution in PBS was run through the flow cell until signal stabilization; then, the surface was rinsed with PBS for 2 min. As a comparison, we used two known methods for modifying the PC chip surface with APTES and with PAA and GA [30,37,50]. ...
Here, we propose and study several types of quartz surface coatings designed for the high-performance sorption of biomolecules and their subsequent detection by a photonic crystal surface mode (PC SM) biosensor. The deposition and sorption of biomolecules are revealed by analyzing changes in the propagation parameters of optical modes on the surface of a photonic crystal (PC). The method makes it possible to measure molecular and cellular affinity interactions in real time by independently recording the values of the angle of total internal reflection and the angle of excitation of the surface wave on the surface of the PC. A series of dextrans with various anchor groups (aldehyde, carboxy, epoxy) suitable for binding with bioligands have been studied. We have carried out comparative experiments with dextrans with other molecular weights. The results confirmed that dextran with a Mw of 500 kDa and anchor epoxy groups have a promising potential as a matrix for the detection of proteins in optical biosensors. The proposed approach would make it possible to enhance the sensitivity of the PC SM biosensor and also permit studying the binding process of low molecular weight molecules in real time.
Background. Despite on the general trend towards decreasing the incidence of newly diagnosed active forms of tuberculosis, the situation with spreading of this disease in Russian Federation remains extremely tense. At the same time, the diagnosis is carried out according to the standard scheme, which takes about a month; another month takes test formulation for drug sensitivity. Thus, the development of new methods for diagnostics and typing of mycobacteria, as well as practice implementation of these developments is an urgent direction. Modern developments in the field of microfluidic technologies open up great opportunities in this direction. Aim. Development of a method for identification and typing of Mycobacterium tuberculosis using a label-free biosensor on surface waves in a one-dimensional photonic crystal (PC SM biosensor). Methods. Oligonucleotide probes were selected and synthesized as DNA targets for M. tuberculosis typing. The photonic crystal surface was modified with aqueous solutions of (3-aminopropyl)triethoxysilane, Leuconostoc mesenteroides dextrans and bovine serum albumin. Experiments were carried out using a PC SM biosensor. Results. Sequences of detecting oligonucleotide probes were selected for spoligotyping of M. tuberculosis on the PC SM biosensor. Modification of their 3'-ends was carried out in order to create extended single-stranded regions that are not subject to the formation of secondary structures and facilitate hybridization with a single-stranded DNA target. Several series of experimental modifications of the PC surface were carried out by using L. mesenteroides dextrans with different functional groups (including detection of the modification results real time) with simultaneous registration of the increment layer size and volume refractive index of the mixture, which excludes the use of a reference cell. Other experiments were carried out to detect the specific binding of biotinylated oligonucleotide probes to the modified PC surface. Conclusions. A technique for the design of probes was developed and a model system of oligonucleotides for the detection of single-stranded DNA using a PC biosensor was proposed. The developed technique of modification of the PC surface with dextrans from L. mesenteroides, which allows to increase the sensitivity of detection of oligonucleotides using the PC SM biosensor. This approach will further expand the panel of diagnostic probes, including identification of resistance markers.
Glutaraldehyde possesses unique characteristics that render it one of the most effective protein crosslinking reagents. It can be present in at least 13 different forms depending on solution conditions such as pH, concentration, temperature, etc. Substantial literature is found concerning the use of glutaraldehyde for protein immobilization, yet there is no agreement about the main reactive species that participates in the crosslinking process because monomeric and polymeric forms are in equilibrium. Glutaraldehyde may react with proteins by several means such as aldol condensation or Michael-type addition, and we show here 8 different reactions for various aqueous forms of this reagent. As a result of these discrepancies and the unique characteristics of each enzyme, crosslinking procedures using glutaraldehyde are largely developed through empirical observation. The choice of the enzyme-glutaraldehyde ratio, as well as their final concentration, is critical because insolubilization of the enzyme must result in minimal distortion of its structure in order to retain catalytic activity. The purpose of this paper is to give an overview of glutaraldehyde as a crosslinking reagent by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.
Guanine-rich DNA/RNA fragments can fold into G-quadruplexes (G4s) – non-canonical four-strand secondary structures. The article contains data on quadruplex interaction with human proteins. Binding of three topologically different G4 structures to more than 9000 human proteins was analyzed. Physicochemical methods were used to verify the results.The dataset was generated to identify the protein targets for DNA quadruplex structures for the purpose of better understanding the role of the structures in gene expression regulation. Presented data include functional interpretation of obtained gene lists, visualized with Cytoscape.
New natural and chemically modified DNA aptamers that inhibit HIV-1 activity at submicromolar concentrations (presumably via preventing viral entry into target cells) are reported. The new DNA aptamers were developed based on known intramolecular G-quadruplexes (G4s) that were functionally unrelated to HIV inhibition [the thrombin-binding aptamer and the fragment of the human oncogene promoter (Bcl2)]. The majority of previously described DNA inhibitors of HIV infection adopt intermolecular structures, and thus their folding variability represents an obvious disadvantage. Intramolecular architectures refold correctly after denaturation and are generally easier to handle. However, whether the G4 topology or other factors account for the anti-HIV activity of our aptamers is unknown. The impact of chemical modification (thiophosphoryl internucleotide linkages) on aptamer activity is discussed. The exact secondary structures of the active compounds and further elucidation of their mechanisms of action hopefully will be the subjects of future studies.
Noncanonically structured DNA aptamers to thrombin were examined. Two different approaches were used to improve stability, binding affinity and biological activity of a known thrombin-binding aptamer. These approaches are chemical modification and the addition of a duplex module to the aptamer core structure. Several chemically modified aptamers and the duplex-bearing ones were all studied under the same conditions by a set of widely known and some relatively new methods. A number of the thrombin-binding aptamer analogs have demonstrated improved characteristics. Most importantly, the study allowed us to compare directly the two approaches to aptamer optimization and to analyze their relative advantages and disadvantages as well as their potential in drug design and fundamental studies.
Thiolated polymers containing disulfide linkages are commonly researched in gene delivery with the assumption that the thiolated complexes form disulfide bonds. This study investigates the extent of disulfide linking in a thiol-containing polymer and determines the impact that free thiols have on the polymer's delivery potential. A fluorescent cationic polymer containing thiol pendant chains was prepared from poly(allylamine) and 2-iminothiolate (Traut's reagent). Polymer fluorescence was determined by UV plate readings and fluorescent microscopy. Transfection efficiency and cytotoxicity were assessed in MCF-7 breast cancer cells. Results show that thiolated polymers exhibited fluorescence at ex/em ∼595/620. Fluorescent measurements, microscopy imaging, and DNA electrophoresis show that thiolated polymers are not internalized by cells in a culture, yet, they bind to the cell surface, perhaps valuable for applications requiring cell adhesion.
The propagation of bound optical waves along the surface of a one-dimensional (1-D) photonic crystal (PC) structure is considered. A unified description of the waves in 1-D PCs for both s- and p-polarizations is done via an impedance approach. A general dispersion relation that is valid for optical surface waves with both polarizations is obtained, and conditions are presented for long-range propagation of plasmon-polariton waves in nanofilms (including lossy ones) deposited on the top of the 1-D PC structure. A method is described for designing 1-D PC structures to fulfill the conditions required for the existence of the surface mode with a particular wavevector at a particular wavelength. It is shown that the propagation length of the long-range surface plasmon-polaritons can be maximized by wavelength tuning, which introduces a slight asymmetry in the system. Comment: 23 pages, 7 figures
The selective adsorption of reagents is often essential for bioarray and lab-on-a-chip type devices. As the starting point for a bioarray, alkyl monolayer terminated silicon shards were photopatterned in a few nanoseconds with thousands of wells (spots) using an optical element, a microlens array. Polyallylamine (PAAm), a primary amine containing polymer, adsorbed with little selectivity to the spots, i.e., silicon oxide, over the hydrophobic background. However, at appropriate concentrations, addition of a cationic surfactant to the PAAm deposition solution, cetyltrimethylammonium chloride, prevented the nonspecific adsorption of PAAm onto the hydrophobic monolayer, while directing it effectively to the active spots on the device. A nonionic surfactant was less effective in preventing the nonspecific adsorption of PAAm onto the hydrophobic monolayer. The localized reactions/interactions of adsorbed PAAm with four species that are useful for bioconjugate chemistry: glutaric anhydride, phenylenediisothiocyanate, biotin NHS ester, and an oligonucleotide (DNA) were shown in the spots of an array. The reactivity of PAAm was further demonstrated with an isocyanate. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) played an important role in confirming selective surface reactivity and adsorption. X-ray photoelectron spectroscopy (XPS), spectroscopic ellipsometry, and wetting confirmed PAAm reactivity on planar substrates.
Protein biochips are at the heart of many medical and bioanalytical applications. Increasing interest has been focused on surface activation and subsequent functionalization strategies for immobilizing these biomolecules. Different approaches using covalent and noncovalent chemistry are reviewed; particular emphasis is placed on the chemical specificity of protein attachment and on retention of protein function. Strategies for creating protein patterns (as opposed to protein arrays) are also outlined. An outlook on promising and challenging future directions for protein biochip research and applications is also offered.
Glutaraldehyde possesses unique characteristics that render it one of the most effective protein crosslinking reagents. It can be present in at least 13 different forms depending on solution conditions such as pH, concentration, temperature, etc. Substantial literature is found concerning the use of glutaraldehyde for protein immobilization, yet there is no agreement about the main reactive species that participates in the crosslinking process because monomeric and polymeric forms are in equilibrium. Glutaraldehyde may react with proteins by several means such as aldol condensation or Michael-type addition, and we show here 8 different reactions for various aqueous forms of this reagent. As a result of these discrepancies and the unique characteristics of each enzyme, crosslinking procedures using glutaraldehyde are largely developed through empirical observation. The choice of the enzyme-glutaraldehyde ratio, as well as their final concentration, is critical because insolubilization of the enzyme must result in minimal distortion of its structure in order to retain catalytic activity. The purpose of this paper is to give an overview of glutaraldehyde as a crosslinking reagent by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.
Microarrays of proteins and peptides make it possible the screening of thousands of binding events in a parallel and high throughput fashion; therefore they are emerging as a powerful tool for proteomics and clinical assays. The complex nature of Proteome, the wide dynamic range of protein concentration in real samples and the critical role of immobilized protein orientation must be taken into account to maximize the utility of protein microarrays. Immobilization strategy and designing of an ideal local chemical environment on the solid surface are both essential for the success of a protein microarray experiment. This review article will focus on protein and peptide arrays highlighting their technical challenges and presenting new directions by means of a set of selected recent applications.
We present a detailed study of synthesis, structural characterization of sharp, spike-like carbon structures and its application for high-resolution atomic-force microscopy (AFM) measurements of biological molecules. The probes are obtained by chemical vapor deposition of spike-like carbon structures on the apexes of common AFM silicon probes. The deposition process is carried out in carbonaceous gas mixture activated by a direct-current discharge. It was revealed by electron microscopy and Raman spectroscopy that the having dimensions at their ends of few nanometers the structures consist of amorphous carbon. The carbon spikes demonstrate high efficiency and resolutions in AFM studies of biological objects. Sub-molecular resolution is demonstrated on the samples of DNA and streptavidin molecules in AFM measurements with the ultra-sharp carbon tips.
An electrochemical method was used to characterize the interaction between Hoechst 33258 and the short DNA sequence related to E. coli lac gene. The probe ssDNA was immobilized on poly(allylamine hydrochloride) via physical adsorption and covalent attachment. The voltammetric signals of Hoechst 33258 have been investigated using screen printed electrode. The interaction of Hoechst 33258 with ssDNA and dsDNA has been studied and proven to have a promising strategy in improving the selectivity of the DNA sensor. The extent of hybridization was evaluated on the basis of the difference between signals of Hoechst 33258 with a probe DNA before and after hybridization with the target ssDNA. Control experiments with non-complementary were performed to test the selectivity of the developed DNA biosensor. The redox current was linearly related to the concentration of the target ssDNA from 1.5-6 μ M with a detection limit of 0.6 μ M.
Polymer nanocapsules have attracted a great deal of interest for drug-delivery and bioimaging applications owing to their functional versatility. The present study focuses on the synthesis, characterisation, biocompatibility and genotoxicity studies of polyallylamine hydrochloride (PAH) nanocapsules for drug-delivery studies. In vitro studies included are haemobiocompatibility studies, cytotoxicity and comet assay in peripheral blood mononuclear cells (PBMCs). Post-intravenous administration of PAH nanocapsules, alteration of haematological parameters, inflammatory marker status, toxicity markers in serum and major organs, RT-PCR, western blotting and histopathological studies of major tissues of rats were evaluated for 30 days. The results of these in vitro studies indicated the biocompatible nature of the PAH nanocapsules at the tested concentrations (1.5 x 10(5) to 6.0 x 10(5) capsules per mL). The activity of in vivo toxicity markers, inflammatory marker status like cyclooxygenase (COX), lipooxygenase (LOX), nitric oxide synthase (NOS) and prostaglandin E2 (PGE2) activity, alteration of haematological parameters and RT-PCR analysis of important genes like interleukin-1 beta (IL-1 beta), monocyte chemotactic protein-1(MCP-1), transforming growth factor beta-1 (TGF-beta 1), kidney injury molecule-1 (Kim-1), heat shock protein gene (Hsp70-1) and tumor necrosis factor alpha (TNF-alpha) showed the least changes. Western blotting studies on immunoregulatory proteins like cytokines (IL-8), chemokines (MIP-2) and cell-adhesion molecules (VCAM-1 and ICAM-1) showed the smallest levels of toxicity with PAH nanocapsule interaction. Histopathological studies of important tissues showed almost normal architecture after treatment with PAH nanocapsules throughout the experimental period. The abovementioned results confirm the biocompatibility and non-toxicity of PAH nanocapsules, thus suggesting their potential for in vivo drug-delivery and bioimaging applications.
Nowadays gene delivery is a topic in many research studies. Non-viral vectors have many advantages over viral vectors in terms of safety, immunogenicity and gene carrying capacity but they suffer from low transfection efficiency and high toxicity. In this study, polyallylamine (PAA), the cationic polymer, has been modified with hydrophobic branches to increase the transfection efficiency of the polymer. Polyallylamine with molecular weights of 15 and 65kDa was selected and grafted with butyl, hexyl and decyl acrylate at percentages of 10, 30 and 50. The ability of the modified polymer to condense DNA was examined by ethidium bromide test. The complex of modified polymer and DNA (polyplex) was characterized for size, zeta potential, transfection efficiency and cytotoxicity in Neuro2A cell lines. The results of ethidium bromide test showed that grafting of PAA decreased its ability for DNA condensation but vectors could still condense DNA at moderate and high carrier to DNA ratios. Most of polyplexes had particle size between 150 and 250nm. The prepared vectors mainly showed positive zeta potential but carriers composed of PAA with high percentage of grafting had negative zeta potential. The best transfection activity was observed in vectors with hexyl acrylate chain. Grafting of polymer reduced its cytotoxicity especially at percentages of 30 and 50. The vectors based of PAA 15kDa had better transfection efficiency than the vectors made of PAA 65kDa. In conclusion, results of the present study indicated that grafting PAA 15kDa with high percentages of hexyl acrylate can help to prepare vectors with better transfection efficiency and less cytotoxicity.
Copyright © 2015 Elsevier B.V. All rights reserved.
We investigated the antimicrobial properties of the cationic polymer polyallylamine (PA) when covalently bonded to glass. The objective was to obtain a robust attachment, yet still allow extension of the polymer chain into solution to enable interaction with the bacteria. The PA film displayed strong antimicrobial activity against Staphylococcus epidermidis , Staphylococcus aureus , and Pseudomonas aeruginosa , which includes both Gram-positive and Gram-negative bacteria. Glass surfaces were prepared by a straightforward two-step procedure of first functionalizing with epoxide groups using 3-glycidoxypropyltrimethoxy silane (GOPTS) and then exposing to PA so that the PA could bind via reaction of a fraction of its amine groups. The surfaces were characterized using X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy to verify the presence of the polymer on the surface, zeta potential measurements to estimate the surface charge of the films, and atomic force microscopy to determine the extension of the polymer chains into solution. Antimicrobial properties of these coatings were evaluated by spraying aqueous suspensions of bacteria on the functionalized glass slides, incubating them under agar, and counting the number of surviving cell colonies.
Highly toxic polyallylamine (PA) was reacted with a varying amount of a novel linker, 6-(N,N,N′,N′-tetramethylguanidinium chloride) hexanoic acid (Tmg-HA), to prepare a series of tetramethylguanidinium-PA (Tmg-PA) polymers, which were used as vectors for gene transfection. The extent of attachment of the linker, Tmg-HA, to the PA backbone was determined by 2,4,6-trinitrobenzene sulfonic acid assay. The modified polymers (Tmg-PAs), when complexed with pDNA, exhibited good condensation ability. The nanoparticles, so formed, were characterized by their size and zeta potential and were subsequently evaluated for their toxicity and transfection ability on various mammalian cells, viz., HeLa, CHO, and HEK 293 cells. Mobility shift assay revealed that on increasing the percent substitution of Tmg-HA onto PA (from Tmg-PA1 to Tmg-PA6), relatively higher amounts of modified polymers were required to retard the mobility of a fixed amount of DNA. Besides, Tmg-PA polymers provided sufficient protection (ca. 84–88%) to bound DNA against nucleases and one of the formulations, Tmg-PA2 (ca. 15% substitution) displayed the highest transfection efficiency outcompeting the commercial transfection reagent, Lipofectamine™ with minimal cytotoxicity. More impressively, the transfection efficiency increased despite recording a decrease in the buffering capacity of the grafted polymers suggesting that buffering capacity is not the sole parameter in determining the gene delivery efficiency of a vector system. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012
Here we show that ionic self-assembly of simple biological tectons can be used to synthesize stable and highly ordered molecular structures. In particular, nucleotides and charged polypeptides can be assembled to form a complex analogous to DNA under relatively benign conditions. The combination of polylysine and pure dGMP leads to a fourfold ladder structure stabilizing an interior G-quartet structure by four polypeptide scaffolds. Making use of the Watson–Crick G∶C base pairing motive leads to double-stranded complexes. Interestingly, these complexes show stable DNA-like organization in aqueous solutions, as proven by gel electrophoresis and intercalation experiments
A straightforward immunoassay based on surface enhanced fluorescence (SEF) has been demonstrated using a fluorescent immune substrate and antibody functionalized-silver nanoparticles. Unlike the conventional SEF-based immunoassay, which usually uses the dye-labeled antibodies and the metallic nanostructured-substrates, the presented immune system does not need the antibodies to be labeled with dye molecules. Thus, this immunoassay can be easily applied to the detection of a wide range of target antigens, which is of great importance for its practical application. The experimental results show that this immunoassay has a good specificity as well as the capacity of quantitative detection. Basically, the surface density of the immuno-adsorbed silver nanoparticles increases with the increased amount of target antigens, resulting in a fluorescence enhancement up to around 7 fold. The dose-responsive performance of the immunoassay has been investigated and the limit of detection (LOD) is 1 ng/mL. Due to its simple preparation method and the wide range of detectable antigens, this presented immunoassay is expected to be helpful for extending the SEF-based application.
Layer-by-layer (LbL) assembly of oppositely charged polyelectrolytes was used to coat fluorescein particles. These particles, with a size of 4−9 μm, were prepared by precipitation of fluorescein at pH 2. Polystyrensulfonate (PSS) and polyallylamine (PAH) were used to form a polyelectrolyte shell on the fluorescein core. The permeation of fluorescein molecules through the polyelectrolyte shell during core dissolution was monitored at pH 8 by the increasing fluorescence intensity as a result of dequenching. The number of polyelectrolyte layers sufficient to sustain fluorescein release was found to be 8−10. Increasing the number of layers prolonged the core dissolution time for minutes. The permeability of polyelectrolyte multilayers of the thickness of 20 nm for fluorescein is about 10-9 m/s. The features of the release profile and possible applications of the LbL method for shell formation in order to control release properties for entrapped materials are outlined.
Coadsorption of polymers and surfactants is a poorly understood process that occurs in a variety of complex fluid applications. In single-component solutions, the cationic polyelectrolyte polylysine and the cationic surfactant cetyltrimethylammonium bromide (CTAB) both adsorb to negatively charged silica surfaces. Here we use scanning angle reflectometry to contrast adsorption from single-component solutions with a sequential adsorption process and a coadsorption process. When adsorbed from single-component solutions, polylysine adsorbs irreversibly, whereas CTAB adsorption is reversible. In the sequential adsorption case, CTAB neither displaces nor adsorbs to preadsorbed polylysine layers. When solutions contain both CTAB and polylysine, they coadsorb to form mixed layers. Mixed layer formation is indicated by a dramatic alteration of the kinetics and reversibility of adsorption compared to either single-component case. The amounts of CTAB and polylysine adsorbed in the mixed layers are both similar to the amounts adsorbed from the respective single-component solution.
The stability of hollow microcapsules against environmental alterations such as pH, osmotic pressure, and temperature is a critical issue for practical applications. It is demonstrated here that multilayer capsules assembled from poly(allylamine hydrochloride) (PAH) and sodium poly(styrene sulfonate) (PSS) can be considerably stabilized by cross-linking of only the PAH component with glutaraldehyde (GA). Formation of a Schiff base between the aldehyde and the amine groups was evidenced by UV−vis spectroscopy. After cross-linking by 2% GA for 2 h, an apparently thicker capsule wall was obtained with higher folds, and no alteration of the macroscopic topology of the capsules was observed after incubation in 0.1 M NaOH for 24 h. The cross-linking significantly improved the mechanical strength of the capsules to resist osmotic pressure induced invagination. Consequently, both the critical pressure and the elasticity modulus (680 MPa) of the capsule wall were doubled compared with that of the control. The cross-linking also greatly lowered the permeability of the capsule wall, as evidenced by confocal laser scanning microscopy and fluorescence recovery after photobleaching. Quantitative analysis revealed that the permeation coefficient for dextran (Mw 250 kD) was reduced by a factor of 3 after cross-linking.
Vascular tissue engineering aims to develop implantable blood-vessels, exhibiting biological and biomechanical characteristics close to those of the native vessels. The ultimate goal of our group is to engineer suitable blood vessel substitutes which could be stored for a long time in vascular bank conditions.First attempts tried to develop coating procedures allowing endothelial cells (EC) differentiation, adhesion and retention on current vascular substitutes but the weak in vivo patency of these grafts was related. Since 2003, our group have been evaluated a new surface modification of internal surface of blood vessels based on polyelectrolyte films coating. The layer-by-layer self-assembly and the resulting polyelectrolyte multiplayer films (PEM) is a simple and versatile way to engineer surfaces with highly specific properties. Previous studies indicated that the poly(sodium-4 styrene sulfonate)/poly (allylamine hydrochloride) PSS/PAH multilayered films when ended by PAH, induce strong adhesion and retention of mature EC which spread and keep their phenotype as well on glass, on expanded polytetrafluoroethylene ePTFE and on cryopreserved arteries. The mechanical properties (compliance), leading to early intimal hyperplasia and graft failure, were lost after artery cryopreservation. We have demonstrated that the compliance and elasticity restoration of PEM treated cryopreserved arteries close to native arteries.In other respect, the use of the circulating progenitor which could be differentiated into matures vascular cell offers new opportunities in vascular engineering. Currents protocols, expend at least 1 month to observe both smooth muscle (SMCs) and endothelium (ECs)-like morphology and about two months for confluent monolayer cells. The progenitor cells cultivated on PEM treated glass showed mature and functional vascular cells (SMCs and ECs) development after only 14 days of culture. The morphological appearance, mature and healthy phenotype markers expression and repartition of differentiated cells are close to mature cells.Challenge now is to build up in less a month, an autologous cellularized vascular graft using patient peripheral stem cells.
Two types of hexaglycylamide (HGA) epitaxial lamellar structures coexisting on the surface of highly oriented pyrolytic graphite (HOPG) exposed to water solutions were studied by high-resolution atomic force microscopy (AFM). Lamellae are distinguished by growth direction and by morphology. The lamellae of the first type (L1) produced by depositions from more dilute solutions are close-packed with a period of ∼5.2 nm, twice the HGA molecular length, and form highly ordered domains morphologically similar to the lamellar domains of alkanes. The less-ordered lamellae of the second type (L2) appear at intermediate and large HGA concentrations and demonstrate variable lamellar width, morphological diversity, and a tendency to merge. The interlamellar separation in the domains of close-packed L2 lamellae varies with the discrete increment ∼2.5 nm; the most frequently observed value is ∼7.5-8.0 nm corresponding to the triple HGA molecular length. The growth directions of lamellae of each type have sixfold rotational symmetry indicating epitaxy with graphite; however, the rosettes of L1 and L2 lamellae orientations are misaligned by 30°. The molecular modeling of possible HGA epitaxial packing arrangements on graphite and their classification have been conducted, and the energetically preferable structures are selected. On this basis, the structural models of the L1 and L2 lamellae are proposed explaining the experimentally observed peculiarities as follows: (1) the L1 and L2 lamellae are respectively parallel and antiparallel β-sheets with two HGA molecules in the unit cell oriented normally to the lamellae boundaries, (2) HGA molecules in L1 and L2 lamellae have different orientations with respect to the graphite lattice, respectively along the directions <1120> and <1010>, (3) L1 lamella is the assembly of two hydrogen-bonded parallel β-sheets oriented head-to-head, (4) L2 lamellae are assemblies of several molecular rows (antiparallel β-sheets) cross-linked by hydrogen bonds. The AFM observations indicate that the covering of the hydrophobic graphite by the dense, closely packed, well-ordered monolayers of hydrophilic oligopeptide is possible.
Protein/surface interactions are well known to play an important role in various biological phenomena and to determine the ultimate biofunctionality of a given material once it is in contact with a biological environment. Control over the interactions between proteins and material surfaces are not only of great theoretical interest but also of crucial importance for many biomedical applications. In this Feature Article, we summarize various successful approaches used in our laboratory and other groups for controlling protein adsorption through chemical modification of the surface and/or the introduction of specific topographic features. Some perspectives on future research in these areas are also presented.
In this study, poly(allylamine hydrochloride) (PAA/HCl) was cross-linked with fermentation bacterial waste (Escherichia coli) in order to introduce a large amount of amine groups as binding sites for potassium hexachloroplatinate(IV), as a model anionic pollutant. The sorption performance of PAA/HCl-modified E. coli was greatly affected by the dosages of PAA/HCl and crosslinker (epichlorohydrin, ECH), and by the pH of the modification reaction medium. These factors were optimized through the response surface methodology (RSM). A three-level factorial Box-Behnken design was performed, and a second-order polynomial model was successfully used to describe the effects of PAA/HCl, ECH and the pH on the Pt(IV) uptake (R(2) = 0.988). The optimal conditions that were obtained from the RSM were 0.49 g of PAA/HCl, 0.05 mL of ECH and pH 10.02, with 1.0 g of dried E. coli biomass. The biosorption isotherm and kinetics studies were carried out in order to evaluate the sorption potential of the PAA/HCl-modified E. coli that was prepared under the optimized conditions. The sorption performance of the developed bacterial biosorbent was 4.36 times greater than that of the raw E. coli. Desorption was carried out using 0.05 M acidified thiourea and the biosorbent was successfully regenerated and reused up to four cycles. Therefore, this simple and cost-effective method suggested here is a useful modification tool for the development of high performance biosorbents for the recovery of anionic precious metals.
A high-precision optical biosensor technique capable of independently determining the refractive index (RI) of liquids is presented. Photonic crystal surface waves were used to detect surface binding events, while an independent registration of the critical angle was used for accurate determination of the liquid RI. This technique was tested using binding of biotin molecules to a streptavidin monolayer at low and high biotin concentrations. The attained baseline noise is 5x10(-13) m/Hz(1/2) for adlayer thickness changes and 9x10(-8) RIU/Hz(1/2) for RI changes.
Optical biosensors have played a key role in the selective recognition of target biomolecules and in biomolecular interaction analysis, providing kinetic data about biological binding events in real time without labeling. The advantages of the label-free concept are the elimination of detrimental effects from labels that may interfere with fundamental interaction and the absence of a time-consuming pretreatment. The disadvantages of all label-free techniques--including the most mature one, surface plasmon resonance (SPR) technique, are a deficient sensitivity to a specific signal and undesirable susceptibilities to non-specific signals, e.g., to the volume effect of refraction index variations. These variations arise from temperature fluctuations and drifts and they are the limiting factor for many state-of-the-art optical biosensors. Here we describe a new optical biosensor technique based on the registration of dual optical s-polarized waves on a photonic crystal surface. The simultaneous registration of two different optical modes from the same surface spot permits the segregation of the volume and the surface signals, while the absence of metal damping permits an increase in the propagation length of the optical surface waves and the sensitivity of the biosensor. The technique was tested with the binding of biotin molecules to a streptavidin monolayer that has been detected with a signal/noise ratio of about 15 at 1 s signal accumulation time. The detection limit is about 20 fg of the analyte on the probed spot of the surface.
Chlamydia species are obligate intracellular bacteria that require growth inside mammalian cells for propagation and survival. As a result, Chlamydia cannot be grown on conventional bacteriological medium. This property makes Chlamydia difficult organisms to grow and maintain in the laboratory. Up until 1965, passage in the yolk sac of the embryonated hen egg was the only way to isolate and propagate the organism. Since then, a tissue culture system has been available that allows easier laboratory culture of the Chlamydia species. However, with the exception of the LGV serovars, most C. trachomatis strains do not readily infect tissue culture cells. Chemical or mechanical assistance is used to increase their infectivity. Today, large numbers of infectious organisms can be purified through Renografin density gradient centrifugation of infected cell lysates. The ability to propagate C. trachomatis in the laboratory has greatly increased the understanding of the pathogenesis of C. trachomatis organisms.
The highly siliceous cell walls of diatoms are probably the most outstanding examples of nanostructured materials in nature. Previous in vitro experiments have shown that the biomolecules found in the cell walls of diatoms, namely polyamines and silaffins, are capable of catalysing the formation of silica nanospheres from silicic/oligosilicic acid solutions. In a previous publication, silica precipitation was found to be strictly correlated with a phosphate-induced microscopic phase separation of the polyamines. The present contribution further characterises the phase separation behaviour of polyamines in aqueous solutions. In particular, a pronounced pH-dependence of the average particle diameter is found. It is, furthermore, shown that the ability of phosphate ions to form polyamine aggregates in aqueous solutions cannot be a purely electrostatic effect. Instead, a defined hydrogen-bonded network stabilised by properly balanced electrostatic interactions should be considered. Finally, solid-state 31P NMR studies on phase-separated polyamines, synthetic silica precipitates, and diatom cell walls from the species Coscinodicus granii support the assumption of a phosphate-induced phase separation process taking place during cell wall formation.
A single layer of poly(allylamine) with a covalently attached osmium pyridine-bipyridine complex adsorbed onto a Au surface modified by mercaptopropanesulfonate has been studied theoretically with a molecular approach and experimentally by cyclic voltammetry. These investigations have been carried out at different pHs and ionic strengths of the electrolyte solution in contact with the redox polyelectrolyte modified electrode. The theory predicts strong coupling between the acid-base and redox equilibria, particularly for low ionic strength, pH close to the pKa, and high concentration of redox sites. The coupling leads to a decrease in the peak potential at pH values above the apparent pKa of the weak polyelectrolyte, in good agreement with the experimental pH dependence at 4 mM NaNO3. Theoretical calculations suggest that the inflection point in the peak position versus pH curves can be used to estimate the apparent pKa of the amino groups in the polymer. Comparison of the apparent pKa for PAH-Os in the film with that of poly(allylamine) reported in the literature shows that the underlying charged thiol strongly influences charge regulation in the film. A systematic study of the film thickness and the degree of protonation in sulfonate and amino groups for solutions of different pH and ionic strength shows the coupling between the different interactions. It is found that the variation of the film properties has a non-monotonic dependence on bulk pH and salt concentration. For example, the film thickness shows a maximum with electrolyte ionic strength, whose origin is attributed to the balance between electrostatic amino-amino repulsions and amino-sulfonate attractions.
- O V Morozova
O.V. Morozova, et al.
International Journal of Adhesion and Adhesives 92 (2019) 125-132
Cultivation and laboratory maintenance of Chlamydia trachomatis
- Scidmore