Bifunctional Cross-linking Agent Research Articles

An Ultrasensitive Voltammetric Genosensor for the Detection of Bacteria Vibrio cholerae in Vegetable and Environmental Water Samples.

In view of the presence of pathogenic Vibrio cholerae (V. cholerae) bacteria in environmental waters, including drinking water, which may pose a potential health risk to humans, an ultrasensitive electrochemical DNA biosensor for rapid detection of V. cholerae DNA in the environmental sample was developed. Silica nanospheres were functionalized with 3-aminopropyltriethoxysilane (APTS) for effective immobilization of the capture probe, and gold nanoparticles were used for acceleration of electron transfer to the electrode surface. The aminated capture probe was immobilized onto the Si-Au nanocomposite-modified carbon screen printed electrode (Si-Au-SPE) via an imine covalent bond with glutaraldehyde (GA), which served as the bifunctional cross-linking agent. The targeted DNA sequence of V. cholerae was monitored via a sandwich DNA hybridization strategy with a pair of DNA probes, which included the capture probe and reporter probe that flanked the complementary DNA (cDNA), and evaluated by differential pulse voltammetry (DPV) in the presence of an anthraquninone redox label. Under optimum sandwich hybridization conditions, the voltammetric genosensor could detect the targeted V. cholerae gene from 1.0 × 10-17-1.0 × 10-7 M cDNA with a limit of detection (LOD) of 1.25 × 10-18 M (i.e., 1.1513 × 10-13 µg/µL) and long-term stability of the DNA biosensor up to 55 days. The electrochemical DNA biosensor was capable of giving a reproducible DPV signal with a relative standard deviation (RSD) of <5.0% (n = 5). Satisfactory recoveries of V. cholerae cDNA concentration from different bacterial strains, river water, and cabbage samples were obtained between 96.5% and 101.6% with the proposed DNA sandwich biosensing procedure. The V. cholerae DNA concentrations determined by the sandwich-type electrochemical genosensor in the environmental samples were correlated to the number of bacterial colonies obtained from standard microbiological procedures (bacterial colony count reference method).

Nanoprobes based on fluorescent semiconductor nanocrystals and single-domain antibodies for highly sensitive detection of epidermal growth factor receptor in tumor cells

Background. The development of highly oriented conjugates of quantum dots (QDs) and single-domain antibodies (sdAbs) as innovative fluorescence imaging nanoprobes that specifically recognize tumor biomarkers, in particular, epidermal growth factor receptor (EGFR), is a promising approach to improving immunohistochemical tumor typing.Aim. The study was aimed at developing fluorescent nanoprobes based on QDs and sdAbs that specifically recognize EGFR, as well as evaluating their functional characteristics (size and optical properties) and functional activity.Materials and methods. QDs were obtained using high-temperature organometallic synthesis and transferred into the aqueous phase by means of stepwise replacement of ligands on the QD surface. The QDs and sdAbs were conjugated in an oriented manner using a bifunctional cross-linking agent. Detailed characteristics of the resulting conjugates were analyzed by the dynamic light scattering and immunoassay methods. Functional activity was assessed on the model human epidermoid carcinoma cells line A431.Results. The QD–sdAb conjugates have been standardized in terms of control parameters determining their functional activity, in particular, hydrodynamic diameter and efficiency of binding with target tumor cells. They are characterized by high dispersity, homogeneity, and specific functional activity towards their molecular target.Conclusion. The results demonstrate the potential use of the designed QD–sdAb conjugates for EGRF detection in immunohistochemical typing of tumor.

Isocyanide-based multi-component reactions for carrier-free and carrier-bound covalent immobilization of enzymes.

Strategies for the covalent immobilization of enzymes depend on the type of functional group selected to perform the coupling reaction, and on the relative importance of selectivity, loading capacity, immobilization time and activity/stability of the resulting immobilized preparation. However, no single strategy is applicable for all covalent immobilization methods or can meet all these criteria, exemplifying the challenge of introducing a versatile process broadly compatible with the residues on the surface of proteins and the functional groups of common linkers. Here, we describe the use of isocyanide-based multi-component reactions for the carrier-bound and carrier-free covalent immobilization of enzymes. Isocyanide-based multi-component reactions can accept a wide variety of functional groups such as epoxy, acid, amine and aldehyde, as well as many commercially available bi-functional linkers, and are therefore suitable for either covalent coupling of enzymes on a solid support or intermolecular cross-linking of enzymes. In this strategy, an isocyanide is directly added to the reaction medium, the enzyme supplies either the exposed amine or carboxylic acid groups, and the support (in carrier-bound immobilization) or the bi-functional cross-linking agent (in carrier-free immobilization) provides another reactive functional group. The protocol offers operational simplicity, high efficiency and a notable reduction in time over alternative strategies, and can be performed by users with expertise in chemistry or biology. The immobilization reactions typically require 1-24 h.

Construction of the lactate-sensing fibremats by confining sensor fluorescent protein of lactate inside nanofibers of the poly(HPMA/DAMA)/ADH-nylon 6 core-shell fibremat.

The development of a new materials platform capable of sustaining the functionality of proteinous sensor molecules over an extended period without being affected by biological contaminants in living systems, such as proteases, is highly demanded. In this study, our primary focus was on fabricating new core-shell fibremats using unique polymer materials, capable of functionalizing encapsulated sensor proteins while resisting the effects of proteases. The core-fibre parts of core-shell fibremats were made using a newly developed post-crosslinkable water-soluble copolymer, poly(2-hydroxypropyl methacrylamide)-co-poly(diacetone methacrylamide), and the bifunctional crosslinking agent, adipic dihydrazide, while the shell layer of the nanofibers was made of nylon 6. Upon encapsulating the lactate-sensor protein eLACCO1.1 at the core-fibre part, the fibremat exhibited a distinct concentration-dependent fluorescence response, with a dynamic range of fluorescence alteration exceeding 1000% over the lactate concentration range of 0 to 100 mM. The estimated dissociation constant from the titration data was comparable to that estimated in a buffer solution. The response remained stable even after 5 cycles and in the presence of proteases. These results indicates that our core-shell fibremat platform could serve as effective immobilizing substrates for various sensor proteins, facilitating continuous and quantitative monitoring of various low-molecular-weight metabolites and catabolites in a variety of biological samples.

STUDY OF THE FEATURES OF GEL FORMATION AND RHEOLOGICAL PROP-ERTIES OF HYDROGELS BASED ON AGAR-AGAR

The article shows studies on the study of the kinetics of gelation and the features of the rheological properties of hydrogels based on agar-agar. It has been found that hydrogels have attracted a lot of attention in the last 50 years due to their exceptional wide range of applications. They have a certain degree of elasticity, very similar to natural fabrics due to the high water content. Within the framework of the literature review, it has been shown that polymer hydrogels are usually obtained by polymerization of water-soluble unsaturated compounds in the presence of a bifunctional cross-linking agent or by structuring pre-prepared reactive prepolymers. The ability of polymer hydrogels to undergo bulk transitions between swollen and collapsed phases under the action of an external medium is one of the most important and universal properties of these materials. It has been shown that polymer hydrogels are known that are used as inert matrices for the controlled release of pre-absorbed biologically active substances. In this case, the release rate of the biologically active substance is determined by the nature and structure of the hydrogel polymer framework. The article studies the kinetics of gelation and the features of the rheological properties of hydrogels based on agar-agar in order to obtain modern biocompatible materials for the medical, cosmetological and pharmaceutical industries. It is shown that in compositions that consist exclusively of agar-agar and water, the shortest time for loss of stickiness and film formation is observed. At the same time, with an increase in the content of agar-agar from 25 to 50 %, one can see the most rapid processes of loss of stickiness and film formation. Compositions with the longest time for loss of stickiness and film formation were determined, which contain 25 % agar-agar and 50 % milk and honey, because they will be characterized by a long diffusion time of active substances to the human body. The scientific novelty of the study is related to the study of the effectiveness of gelation in conditions of highly concentrated agar-agar solutions with different contents of the polymer part and active substances, on the basis of which compositions for the preparation of face masks and medical tapes are proposed.

Exploration of a three-dimensional matrix as micro-reactor in the form of reactive polyaminosaccharide hydrogel beads using multipoint covalent interaction approach.

Diversity in backbone polymer composition makes hydrogel-based resources open to broad spectrum of applications. Biomacromolecules which have reactive functional groups in their structural frame and can also exhibit hydrogel properties could be utilized in biomedical, pharmaceutical and drug delivery applications after some chemical modifications. Present study aims towards development of chitosan-based hydrogel system crosslinked together with glucosyltransferase. Hydrogel structure worked as an immobilization matrix and as a microreactor system to catalyze the cleavage of a disaccharide. Uniform chitosan hydrogel beads were prepared and dextransucrase was attached using multipoint covalent interaction approach. Strong interaction was developed by linking polymeric hydrogel with the biocatalyst utilizing glutaraldehyde as spacer arms. This bifunctional crosslinking agent performed two important tasks that includes functionalization of hydrogel beads and crosslinking of this activated matrix system with enzyme fragments. Hydrogel beads required 18.0h crosslinking time with enzyme (6.5mgml-1, 189.9 DSU) under specific environment (4°C, 100rpm) to saturate all available ends. Enzyme fragments were observed bound with hydrogel beads when screened for surface topology indicating successful crosslinking. Steady state kinetics of crosslinked dextransucrase was studied in detail and it was revealed that it can catalyse sucrose in 30.0min at 35°C (pH 5.5) with an energy of activation around 15.23kJmol-1 with increased Vmax (785 DSU ml-1) and Km (256mM) values as compared to soluble enzyme version. Thermal stability of the crosslinked dextransucrase also particularly improved 2.5 fold at 45°C in comparison with soluble enzyme. Improved catalytic performance suggests that multipoint covalent immobilization protocol adapted using hydrogel system could be tailored as microreactor for catalysis of profitable macromolecules.

Supercritical carbon dioxide as a tool for improving the biocompatible properties of biopolymer and tissue specific scaffolds for tissue engineering

Objective: to investigate the efficacy of supercritical carbon dioxide (sc-CO2) for enhancштп the biocompatibility of biopolymer scaffolds from biodegradable materials and tissue-specific scaffolds from decellularized porcine liver slices (PLSs) or fine porcine cartilage particles (FPCPs).Materials and methods. Biopolymer scaffolds of a polyoxy(butyrate-co-valerate) and gelatin copolymer composition, 4 mm in diameter and 80 mm in length, were formed by electrospinning (NANON-01A, MECC CO, Japan) and stabilized by incubation in glutaraldehyde vapor for 48 hours at room temperature. For decellularization, PLSs and FPCPs were incubated under periodic stirring in buffer (pH = 7.4) solutions of sodium dodecyl sulfate (0.1%) and Triton X-100 with increasing concentrations (1, 2, and 3%). Treatment in a sc-CO2 atmosphere was done at 150–300 bar pressure, 35 °C temperature, and 0.25–2.5 mL/min flow rate of sc-CO2 for 8–24 hours. 10% ethanol was introduced as a polarity modifier. Cytotoxicity was studied according to GOST ISO 10993-5-2011. The growth of NIH/3T3 in the presence of samples was studied using an interactive optical system IncuCyte Zoom.Results. The effect of the sc-CO2 flow rate and pressure, and the effect of addition of ethanol, on the biocompatibility of scaffolds was investigated. It was found that treatment at a low sc-CO2 flow rate (0.25 mL/min) does not achieve the required cytotoxicity. Complete absence of cytotoxicity in biopolymer scaffolds was achieved in the presence of 10% ethanol, at a sc-CO2 flow rate of 2.5 mL/min, 300 bar pressure and 35 °C temperature after 8 hours of treatment. Effective removal of cytotoxic detergents from decellularized liver occurs already at a 150-bar pressure and does not require the addition of ethanol. Adding ethanol to sc-CO2 eliminates not only the cytotoxic, but also the cytostatic effect of tissue-specific scaffolds.Conclusion. Sc-CO2 treatment is an effective way to enhance the biocompatibility of three-dimensional porous matrices produced using cytotoxic substances: bifunctional crosslinking agents for biopolymer scaffolds and surfactants in the case of tissue-specific matrices. Addition of ethanol as a polarity modifier improves the treatment efficiency by eliminating both cytotoxic and cytostatic effects.

Preparation and characterization of HBc virus like particles with site-directed coupling function

Hepatitis B virus core protein can self-assemble into icosahedral symmetrical viral-like particles (VLPs) in vitro, and display exogenous sequences repeatedly and densely on the surface. VLPs also have strong immunogenicity and biological activity. When the nanoparticles enter the body, they quickly induce specific humoral and cellular immune responses to exogenous antigens. In this study, we designed an HBc-VLPs that can be coupled with antigens at specific sites, and developed a set of efficient methods to prepare HBc-VLPs. Through site-specific mutation technology, the 80th amino acid of peptide was changed from Ala to Cys, a specific cross-linking site was inserted into the main immunodominant region of HBc-VLPs, and the prokaryotic expression vector pET28a(+)-hbc was constructed. After expression and purification, high purity HBc(A80C) monomer protein was assembled into HBc-VLPs nanoparticles in Phosphate Buffer. The results of particle size analysis show that the average particle size of nanoparticles was 29.8 nm. Transmission electron microscopy (TEM) showed that HBc-VLPs formed spherical particles with a particle size of about 30 nm, and its morphology was similar to that of natural HBV particles. The influenza virus antigen M2e peptide as model antigen was connected to Cys residue of HBc-VLPs by Sulfo-SMCC, an amino sulfhydryl bifunctional cross-linking agent, and M2e-HBc-VLPs model vaccine was prepared. The integrity of HBc-VLPs structure and the correct cross-linking of M2e were verified by cell fluorescence tracing. Animal immune experiments showed that the vaccine can effectively stimulate the production of antigen-specific IgG antibody in mice, which verified the effectiveness of the vaccine carrier HBc-VLPs. This study lays a foundation for the research of HBc-VLPs as vaccine vector, and help to promote the development of HBc-VLPs vaccine and the application of HBc-VLPs in other fields.

Fabrication of silica-based chitosan biocomposite material from volcanic ash and shrimp husk by sol gel method for adsorbent of cadmium (II) Ions

The utilization of volcanic ash and shrimp shell waste into composite material is very interesting to discuss as a strategy to make naturally available materials into valuable, stable and low cost adsorbent. The study is aimed to fabricate the silica-based chitosan biocomposite material from volcanic ash of Sinabung mountain and shrimp husk by sol gel method to be used as an adsorbent. The procedure is performed by converting volcanic ash into silica salt, extracting the chitosan from shrimp skeleton, and the synthesis of the silica-chitosan biocomposite by mixing the silica with chitosan in the aid of bifunctional glutaraldehyde cross-linking agent. The silica-based chitosan composite is successfully prepared from the mixture of 20 mL 58% (v/v) Na2SiO3 with 2% (w/v) chitosan to obtain biocomposite material (SChC1). A rapid and simple method to bind the silica with chitosan is achieved. The resulting biocomposite is amorphous, in the form of small crystals, a rough surface with a large pore area between 2θ = 20-24°. Adsorption capacity of the biocomposite material has been evaluated, where it has the capability to bind cadmium ion due to the presence of amine group in the material surface. The prepared material of silica-chitosan biocomposite has the potential to be used as an adsorbent.

HERBICIDE GENOTOXICITY REVEALED WITH THE SOMATIC WING SPOT ASSAY OF Drosophila melanogaster

The genotoxicity of the herbicides clomazone, linuron and simazine was detected using the somatic wing spot assay of Drosophila melanogaster . For the evaluation of the mutagenic and recombinogenic activities induced by the herbicides two crosses were used: the standard cross (ST) which expresses basal levels of cytochrome P450 enzymes and the high bioactivation cross (HB) with overexpression of P450 genes conferring increased sensitivity to promutagens and procarcinogens. Third-instar larvae were exposed by chronic feeding (48 h) to three different concentrations of each herbicide. The frequencies of spots per individual in the treated series were compared to the negative concurrent control series (water or 5 % ethanol solution depending on the herbicide). Clomazone showed positive results for small and total spots in both the ST and the HB crosses. Linuron was positive at all concentrations tested for small and total spots in the ST cross while only positive for small and total spots at the highest concentration assayed in the HB cross. Simazine did not produce positive results in the ST cross while it was positive in the HB cross. The bifunctional cross-linking agent mitomycin C (MMC – 0.15 mM) was used as positive control and produced as expected a significant increase of all types of spots in both crosses. In conclusion, the positive results were due to induced mutations and not by recombinogenic activity, furthermore the significant increase in the frequency of total spots was mainly due to the induction of small single spots.

Room-temperature solution-processed, ZrOx-based hybrid gate dielectrics for low-voltage organic thin-film transistors on plastic substrates

For advanced electronic device applications including printable, flexible organic electronics and bioelectronics, it is crucial to develop novel dielectric materials with high dielectric strength and low-temperature processability compatible with plastic substrates for producing flexible and low-voltage organic thin-film transistors (OTFTs). Herein, we report the facile fabrication of rationally designed, high-capacitance hybrid gate dielectric films for low-voltage OTFTs on plastic substrates via a room-temperature solution process. To create room-temperature-processed gate dielectrics with high dielectric strength, we designed organic-inorganic hybrid gate dielectric films by including ZrOx networks as inorganic high-k oxide materials for obtaining large capacitance and 1,6-hexanediol diacrylate as a bifunctional UV cross-linking agent and a flexible organic component for increasing the density and flexibility of films and ensuring room-temperature processability. The UV cross-linked ZrOx-based hybrid composite films with a 15 nm thickness showed excellent dielectric properties with a leakage current density of ∼10−6 A/cm2 and a capacitance of 640 nF/cm2. The film surfaces were modified with hydrophobic self-assembled monolayers to match the surface energy of organic semiconductors for enhancing the OTFT performance. The utility of the hybrid dielectrics for flexible OTFTs was demonstrated by realizing pentacene TFTs on plastic substrates with a low operating voltage at –2.5 V, a high on/off current ratio of 105, a threshold voltage as low as –0.7 V, and a hole mobility up to 0.47 cm2 V−1 s−1, which are comparable to those performed on Si substrates.

The effect of geometric isomerism on the anticancer activity of the monofunctional platinum complex trans-[Pt(NH3)2(phenanthridine)Cl]NO3.

A trans-DDP based monofunctional phenanthridine Pt(ii) complex was synthesized and characterized. Its anticancer activity was studied in vitro on a panel of human cancer cell lines and mouse intestinal cancer organoids. This complex displays significant antitumor properties, with a different spectrum of activity than that of classic bifunctional cross-linking agents like cisplatin.

Improving the performance of immobilized β-glucosidase using a microreactor

Here, we have presented a technically simple and efficient method for preparing a continuous flow microreactor by employing immobilized β-glucosidase in a silica quartz capillary tube. Developing an immobilized enzyme layer on the inner wall of the capillary tube involved the modification of the inner wall using bifunctional crosslinking agents 3-aminopropyltriethoxysilane and glutaraldehyde before attaching β-glucosidase. The microreactor afforded unique reaction capacities compared with conventional batch operational configurations. These included enhanced pH and thermal stability during storage tests, increased conversion rates of cellobiose, and reduced product inhibition. The maximum conversion rate of soluble substrate cellobiose digestion in the microreactor was 76% at 50°C and pH 4.8 when the microreactor was operated continually over 10h at a flow rate of 7μL/min. This was markedly contrasting to the observed conversion rate of 56% when cellobiose was digested in a conventional batch mode under the same pH and temperature conditions. Reaction inhibition by glucose was significantly reduced in the microreactor. We postulate that the increased capacity of glucose to diffuse into the continual flowing media above the immobilized enzyme layer prevents glucose from reaching inhibitory concentrations at the substrate-enzyme interface.

48 Transferrin-modified self-assembled HSA (human serum albumin) nanoparticles enhance drug delivery to tumors

Objectives Taxanes like paclitaxel (Tax), docetaxel and cabazitaxel are effective chemotherapeutic drugs which have been used in various types of cancer in recent years. Taxanes are highly lipophilic and practically insoluble in water. In addition, their clinical applications are limited by their toxicity to normal tissues. Human serum albumin (HSA) nanoparticles (HSA-Nps) have been shown to be a promising drug delivery system. They act by enhancing the drug’s bioavailability in tumors via SPARC. To further improve the targeting efficiency of HSA-Nps, transferrin (Tf) was covalently coupled to the HSA-Nps using NHS-PEG2000-MAL as a bifunctional cross-linking agent, since transferrin receptor is highly expressed in most tumor cells. Methods HSA-Nps encapsulating paclitaxel (HSA-Nps-Tax) were obtained using the salting-out method. The cross-linking agent was then added to react with the amino groups of HSA-Nps. Meanwhile, sulfhydryl groups were introduced to Tf by 2-iminothiolane, which reacts with the amino groups of transferrin. In the third step, sulfhydryl-reactive transferrin was covalently coupled to the HSA-Nps. Results Tf-modified HSA-Nps-Tax (Tf-HSA-Nps-Tax) had an excellent mean particle size (180 nm) and stability, and had greater cytotoxicity and apoptosis-inducing activity in MCF-7 cells than HSA-Nps-Tax in vitro. Furthermore, Tf-HSA-Nps-Tax showed greater tumor growth inhibition than HSA-Nps-Tax in vivo, which highlighted the advantage of transferrin targeting of tumor cells. Conclusions Tf-modified self-assembled HSA Nps are promising targeted carriers for tumor therapeutics. Acknowledgments This research was financially supported by Jilin Province Science and Technology Development Program (Grant Nos. 20140311072YY and 20150520141JH).

Selective disulfide reduction for labeling and enhancement of Fab antibody fragments

Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab’)2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab’)2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications.

Interpenetrating anion exchange membranes using poly(1-vinylimidazole) as bifunctional crosslinker for fuel cells

There is a balance between ionic conductivity and dimensional stability in anion exchange membranes (AEMs). High conductivity resulting from densely charged groups is normally accompanied with high water uptake and excessive swelling. Herein, a series of novel AEMs with interpenetrating network was prepared by the incorporation of poly(vinylbenzyl chloride) (PVBC) and poly(1-vinylimidazole)(PVIm) into the PVA matrix. PVBC and PVIm act as bifunctional macromolecular crosslinking agents to provide both imidazolium cations and to form crosslinked network in the membranes. The resulting AEMs were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). The effect of crosslinking density on conductivity, water uptake, swelling ratio and stability was investigated. The results reveal that the addition of PVBC-c-PVIm could be simultaneously conducive to higher conductivity and lower swelling ratio. Notably, the membrane containing 50wt% PVBC-c-PVIm had IEC of 1.86meqg−1, water uptake of 39.4%, and hydroxide conductivity of 21.9mScm−1 at 30°C. A maximum power density of 37.1mWcm−2 is achieved at a current density of 64mAcm−2. The crosslinked AEMs also exhibit excellent alkaline stability in a 4M KOH solution at 60°C for 1320h.

Synchrotron Small-Angle X-ray Scattering Study of Cross-Linked Polymeric Micelles.

Polymeric micelles of methoxypoly(ethylene glycol)-b-poly(lactide) containing lysine units (mPEG-PLA-Lys4) were cross-linked by reacting of lysine moieties with a bifunctional bis(N-hydroxy-succinimide ester). The micelles were characterized in aqueous solution using dynamic light scattering, transmission electron microscopy, and synchrotron small-angle X-ray scattering. The mPEG-PLA-Lys4 was synthesized through the ring-opening polymerization of N6-carbobenzyloxy-L-lysine N-carboxyanhydride with amine-terminated mPEG-PLA and subsequent deprotection. The polymeric micelles showed enhanced micelle stability after cross-linking, which was confirmed by adding sodium dodecyl sulfate as a destabilizing agent. The average diameters measured via dynamic light scattering were 19.1 nm and 29.2 nm for non-cross-linked polymeric micelles (NCPMs) and cross-linked polymeric micelles (CPMs), respectively. The transmission electron microscopy images showed that the size of the polymeric micelles increased slightly due to cross-linking, which was in good agreement with the DLS measurements. The overall structures and internal structural changes of NCPMs and CPMs in aqueous solution were studied in detail using synchrotron X-ray scattering method. According to the structural parameters of X-ray scattering analysis, CPMs with a more densely packed core structure were formed by reacting bifunctional cross-linking agents with lysine amino groups located in the innermost core of the polymeric micelles.

Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking

The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase.

Cryostructuring of polymeric systems. 38. The causes of the covalently-crosslinked cryogels formation upon the homopolymerization of N,N-dimethylacrylamide in moderately-frozen aqueous media

The redox-initiated homopolymerization of N,N-dimethylacrylamide in a moderately-frozen aqueous medium yields cross-linked high-swelling cryogels instead of soluble polymers that are formed in water solutions at positive temperatures or both at positive and negative temperatures in formamide media. The comparison of swelling characteristics of the cryogels and especially synthesized cross-linked model hydrogels showed that the amount of crosslinks in the cryogels thus prepared was rather small, no more than 0.05–0.10mol.% with respect to the total quantity of monomer units. The studies performed with NMR and EPR spectroscopies revealed that the crosslinks in such cryogels were formed owing to the generation of certain unexpected by-radicals via coupling of the initiating sulfate-anion-radical with the monomer at the carbonyl group of N,N-dimethylacrylamide. These radicals acted as bifunctional cross-linking agents.

Bifunctional Crosslinking Agents Enhance Anion Exchange Membrane Efficacy for Vanadium Redox Flow Batteries.

A series of cross-linked fluorinated poly (aryl ether oxadiazole) membranes (FPAEOM) derivatized with imidazolium groups were prepared. Poly (N-vinylimidazole) (PVI) was used as the bifunctional cross-linking agent to: a) lower vanadium permeability, b) enhance dimensional stability, and c) concomitantly provide added ion exchange capacity in the resultant anion exchange membranes. At a molar ratio of PVI to FPAEOM of 1.5, the resultant membrane (FPAEOM-1.5 PVI) had an ion exchange capacity of 2.2 meq g-1, a vanadium permeability of 6.8×10-7 cm2 min-1, a water uptake of 68 wt.%, and an ionic conductivity of 22.0 mS cm-1, all at 25°C. Single cells prepared with the FPAEOM-1.5 PVI membrane exhibited a higher coulombic efficiency (> 92%) and energy efficiency (> 86%) after 40 test cycles in vanadium redox flow battery. The imidazolium cation showed high chemical stability in highly acidic and oxidizing vanadium solution as opposed to poor stability in alkaline solutions. Based on our DFT studies, this was attributed to the lower HOMO energy (-7.265 eV) of the HSO4- ion (compared to the OH- ion; -5.496 eV) and the larger HOMO-LUMO energy gap (6.394 eV) of dimethylimidazolium bisulfate ([DMIM] [HSO4]) as compared to [DMIM] [OH] (5.387 eV).