Biorelated Polyelectrolyte Coatings Studied by in Situ Attenuated Total Reflection–Fourier Transform Infrared Spectroscopy: Deposition Concepts, Wet Adhesiveness, and Biomedical Applications

In this conceptual contribution, thin functional coatings consisting of either pure polyelectrolytes (PELs) or complexes between oppositely charged PELs at model and applied substrates are outlined. Latter PEL/PEL complexes were deposited by two concepts. In a first well-known concept, PEL multilayers (PEM) were consecutively deposited according to the layer-by-layer (LbL) technique. In a second less known concept, PEL complex (PEC) nanoparticles (NPs) preformed by mixing polycation (PC) and polyanion (PA) solutions were deposited in one step. Both concepts based on binary oppositely charged PELs are compared to one based on a single polycation system. Examples shall be given on adhesiveness, nanostructure, and biomedical applications of PEM and PEC NP coatings. In situ attenuated total reflection (ATR) infrared (IR) spectroscopy, circular dichroism (CD), and scanning force microscopy (SFM) were used for molecular, optical, and microscopic characterization. At first, results on the adsorbed amount and wet adhesiveness of pure (single-component) PEL coatings as a function of charge density are given to motivate coatings of mixed oppositely charged PELs. Second, the wet adhesiveness of PEM and PEC NP coatings of identical PEL compounds in aqueous media varying the molar charge ratio (<i>n</i>–/<i>n</i>+) and the deposition step <i>z</i>, respectively, is compared. Upon comparing the three PEL deposition concepts, it is suggested that the lack or absence of excess charge at the PEL/surface interface is one of the main factors for the wet adhesiveness of all pure PEL, PEM, and PEC NP coatings. Finally, the potential of PEM and PEC NP coatings for biomedical applications is outlined. Concerning biopassivation, PEM coatings excessed or terminated by PA repel proteins with low isoelectric points. Concerning bioactivation, PEM coatings loaded with antibiotics as well as PEC NP coatings loaded with therapeutic bisphosphonates showed retarded, optionally temperature responsive drug release for applications in acute surgery and bone healing, and immunoglobulin/PEL complex coatings might open theranostic applications

Quantitative Proteomics Using Ultralow Flow Capillary Electrophoresis–Mass Spectrometry

In this work, we evaluate the incorporation of an ultralow flow interface for coupling capillary electrophoresis (CE) and mass spectrometry (MS), in combination with reversed-phase high-pressure liquid chromatography (HPLC) fractionation as an alternate workflow for quantitative proteomics. Proteins, extracted from a SILAC (stable isotope labeling by amino acids in cell culture) labeled and an unlabeled yeast strain were mixed and digested enzymatically in solution. The resulting peptides were fractionated using RP-HPLC and analyzed by CE–MS yielding a total of 28 538 quantified peptides that correspond to 3 272 quantified proteins. CE–MS analysis was performed using a neutral capillary coating, providing the highest separation efficiency at ultralow flow conditions (<10 nL/min). Moreover, we were able to demonstrate that CE–MS is a powerful method for the identification of low-abundance modified peptides within the same sample. Without any further enrichment strategies, we succeeded in quantifying 1 371 phosphopeptides present in the CE–MS data set and found 49 phosphopeptides to be differentially regulated in the two yeast strains. Including acetylation, phosphorylation, deamidation, and oxidized forms, a total of 8 106 modified peptides could be identified in addition to 33 854 unique peptide sequences found. The work presented here shows the first quantitative proteomics approach that combines SILAC labeling with CE–MS analysis

Enhanced Activity of Acetyl CoA Synthetase Adsorbed on Smart Microgel: an Implication for Precursor Biosynthesis

Acetyl coenzyme A (acetyl CoA) is an essential precursor molecule for synthesis of metabolites such as the polyketide-based drugs (tetracycline, mitharamycin, Zocor, etc.) fats, lipids, and cholesterol. Acetyl CoA synthetase (Acs) is one of the enzymes that catalyzes acetyl CoA synthesis, and this enzyme is essentially employed for continuous supply of the acetyl CoA for the production of these metabolites. To achieve reusable and a more robust entity of the enzyme, we carried out the immobilization of Acs on poly­(<i>N</i>-isopropylacrylamide)-poly­(ethylenimine) (PNIPAm-PEI) microgels via adsorption. Cationic PNIPAm-PEI microgel was synthesized by one-step graft copolymerization of NIPAm and <i>N</i>,<i>N</i>-methylene bis-acrylamide (MBA) from PEI. Adsorption studies of Acs on microgel indicated high binding of enzymes, with a maximum binding capacity of 286 μg/mg of microgel for Acs was achieved. The immobilized enzymes showed improved biocatalytic efficiency over free enzymes, beside this, the reaction parameters and circular dichroism (CD) spectroscopy studies indicated no significant changes in the enzyme structure after immobilization. This thoroughly characterized enzyme bioconjugate was further immobilized on an ultrathin membrane to assess the same reaction in flow through condition. Bioconjugate was covalently immobilized on a thin layer of preformed microgel support upon polyethylene terephthalate (PET) track etched membrane. The prepared membrane was used in a dead end filtration device to monitor the bioconversion efficiency and operational stability of cross-linked bioconjugate. The membrane reactor showed consistent operational stability and maintained >70% of initial activity after 7 consecutive operation cycles

Bienzymatic Sequential Reaction on Microgel Particles and Their Cofactor Dependent Applications

We report, the preparation and characterization of bioconjugates, wherein enzymes pyruvate kinase (Pk) and l-lactic dehydrogenase (Ldh) were covalently bound to poly­(<i>N</i>-isopropylacrylamide)-poly­(ethylenimine) (PNIPAm-PEI) microgel support using glutaraldehyde (GA) as the cross-linker. The effects of different arrangements of enzymes on the microgels were investigated for the enzymatic behavior and to obtain maximum Pk-Ldh sequential reaction. The dual enzyme bioconjugates prepared by simultaneous addition of both the enzymes immobilized on the same microgel particles (PL), and PiLi, that is, dual enzyme bioconjugate obtained by combining single-enzyme bioconjugates (immobilized pyruvate kinase (Pi) and immobilized lactate dehydrogenase (Li)), were used to study the effect of the assembly of dual enzymes systems on the microgels. The kinetic parameters (<i>K</i>_m, <i>k</i>_cat), reaction parameters (temperature, pH), stability (thermal and storage), and cofactor dependent applications were studied for the dual enzymes conjugates. The kinetic results indicated an improved turn over number (<i>k</i>_cat) for PL, while the <i>k</i>_cat and catalytic efficiency was significantly decreased in case of PiLi. For cofactor dependent application, in which the ability of ADP monitoring and ATP synthesis by the conjugates were studied, the activity of PL was found to be nearly 2-fold better than that of PiLi. These results indicated that the influence of spacing between the enzymes is an important factor in optimization of multienzyme immobilization on the support

Facile Approach to Grafting of Poly(2-oxazoline) Brushes on Macroscopic Surfaces and Applications Thereof

This study reports on a facile and versatile approach for modification of macroscopic surface via grafting of multifunctional poly­(2-oxazoline) molecules in brush-like conformation. For this purpose, carboxyl-terminated poly­(2-isopropyl-2-oxazoline) molecules have been synthesized by ring-opening cationic polymerization and subsequently grafted on underlined substrates by exploiting the ”grafting to” approach. A systematic variation in thickness of the grafted poly (2-isopropyl-2-oxazoline) brushes has been demonstrated. Polymer-modified surfaces have been characterized by means of a number of analytical tools including ellipsometry, X-ray photoelectron spectroscopy, ultraviolate spectroscopy, attenuated total reflection infrared spectroscopy and atomic force microscopy. Interestingly, poly­(2-isopropyl-2-oxazoline) molecules have been found to retain their physical properties even after grafting on macroscopic surfaces. Finally, fabricated polymer brushes have been used as platform for stabilization of inorganic nanoparticles on macroscopic surfaces

Biohybrid Networks of Selectively Desulfated Glycosaminoglycans for Tunable Growth Factor Delivery

Sulfation patterns of glycosaminoglycans (GAG) govern the electrostatic complexation of biomolecules and thus allow for modulating the release profiles of growth factors from GAG-based hydrogels. To explore options related to this, selectively desulfated heparin derivatives were prepared, thoroughly characterized, and covalently converted with star-shaped poly­(ethylene glycol) into binary polymer networks. The impact of the GAG sulfation pattern on the network characteristics of the obtained hydrogels was theoretically evaluated by mean field methods and experimentally analyzed by rheometry and swelling measurements. Sulfation-dependent differences of reactivity and miscibility of the heparin derivatives were shown to determine network formation. A theory-based design concept for customizing growth factor affinity and physical characteristics was introduced and validated by quantifying the release of fibroblast growth factor 2 from a set of biohybrid gels. The resulting new class of cell-instructive polymer matrices with tunable GAG sulfation will be instrumental for multiple applications in biotechnology and medicine

Tunable Strain in Magnetoelectric ZnO Microrod Composite Interfaces

The intrinsic strain at coupled components in magnetoelectric composites plays an important role for the properties and function of these materials. In this in situ X-ray nanodiffraction experiment, the coating-induced as well as the magnetic-field-induced strain at the coupled interface of complex magnetoelectric microcomposites were investigated. These consist of piezoelectric ZnO microrods coated with an amorphous layer of magnetostrictive (Fe₉₀Co₁₀)₇₈Si₁₂B₁₀. While the intrinsic strain is in the range of 10^–4, the magnetic-field-induced strain is within 10^–5, one order of magnitude smaller. Additionally, the strain relaxation distance of around 5 μm for both kinds of strain superposes indicating a correlation. The value of both intrinsic and magnetic-field-induced strain can be manipulated by the diameter of the rodlike composite. The intrinsic interface strain within the ZnO increases exponentially by decreasing the rod diameter while the magnetic-field-induced strain increases linearly within the given range. This study shows that miniaturizing has a huge impact on magnetoelectric composite properties, resulting in a strongly enhanced strain field and magnetic response