Cucurbituril‐mediated immobilization of fluorescent proteins on supramolecular biomaterials

The reversible introduction of functionality at material surfaces is of interest for the development of functional biomaterials. In particular, the use of supramolecular immobilization strategies facilitates mild reaction and processing conditions, as compared to other covalent analogues. Here, the engineering of multicomponent supramolecular materials, beyond the use of a single supramolecular entity is proposed. Cucurbit[8]uril (Q8) mediated host–guest chemistry is combined with hydrogen bonding supramolecular 2‐ureido‐4‐pyrimidinone (UPy)‐based materials. The modular incorporation of a UPy‐additive that presents one guest to incorporate into the Q8 host allows for selective supramolecular functionalization at the water–polymer material interface. Supramolecular ternary complex formation at the material surface was studied by X‐ray photoelectron spectroscopy, which as a result of large overlap in atomic composition of the different components showed minor changes is surface composition upon complex formation. Surface MALDI‐ToF MS measurements revealed useful insights in the formation of complexes. Protein immobilization was monitored using both fluorescence spectroscopy and quartz crystal microbalance with dissipation monitoring, which successfully demonstrated ternary complex formation. Although proteins could selectively be immobilized onto the surfaces, control of the system's stability remains a challenge as a result of the dynamicity of the host–guest assembly. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 3607–3616     

Purification of angiotensin‐converting enzyme from human plasma and investigation of the effect of some active ingredients isolated from Nigella sativa L. extract on the enzyme activity

In the present study, one‐step purification of angiotensin‐converting enzyme (ACE, peptidyldipeptidase A, EC 3.4.15.1), responsible for regulation of blood pressure, was achieved using affinity chromatography from human plasma. The enzyme was purified 12,860‐fold with a specific activtiy of 5080 EU/mg protein. Optimum temperature and pH were determined for the enzyme as 35–40°C and pH 7.4–7.5, respectively. The purity of ACE was determined by SDS–PAGE and the enzyme showed two bands at 60 and 70 kDa on the gel. The native molecular weight of ACE was found to be 260 kDa by gel filtration chromatography, demonstrating that the enzyme has a heterodimeric structure. Natural fatty acids of Nigella sativa (Ranunculaceae) were isolated by means of column chromatography. The structures of these compounds were determined using NMR and GC‐MS. The results showed that high concentrations of linoleic, oleic and palmitic acids were isolated from the plant. The effect of six fractions (Fr 1–6) on ACE activity was examined. Fraction 3 increased the ACE activity while the other fractions decreased the enzyme activity. The concentrations of the fractions inhibiting the half‐maximum activity of the enzyme were calculated as 1.597 mg/mL for Fr 1, 0.053 mg/mL for Fr 2, 0.527 mg/mL for Fr 4, 0.044 mg/mL for Fr 5 and 0.136 mg/mL for Fr 6 using a Lineweaver–Burk graph.     

Synthesis and structural characterization of new bioactive ligands and their Bi(III) derivatives

Five new carboxylic acid precursors bearing thiourea group and their corresponding bismuth(III) complexes were synthesized and characterized using CHNS and inductively coupled plasma analyses and infrared and NMR (¹H, ¹³C) spectroscopies. Single‐crystal X‐ray diffraction analysis was also carried out for one of the precursors. The behaviour of the compounds was bioassayed for antibacterial, antifungal, antioxidant and enzyme (lipoxygenase, α‐glycosidase and anti‐urease) inhibition activities. It is concluded that the interaction of the compounds with bismuth enhances both the antimicrobial and enzyme inhibition activities. The synthesized compounds may prove to be good therapeutic agents.     

Immobilization of Antibodies on Magnetic Carbonaceous Microspheres for Selective Enrichment of Lysine‐acetylated Proteins and Peptides

Lysine acetylation is a dynamic and reversible modification, which has been proved to be a key posttranslational modification in cellular regulation. However, the low amounts of the acetylated proteins could hardly be detected before enrichment. In this study, for the first time, antibody‐immobilized magnetic carbonaceous microspheres were developed for selective enrichment of acetylated proteins and peptides. At first, standard proteins composed of acetylated bovine serum albumin, myoglobin, α‐casein and ovalbumin were used as model proteins to verify the enrichment efficiency. Then, the synthesized peptide was employed to confirm the selectivity of the method. Besides, the antibody‐immobilized magnetic particles were successfully applied to analyze mouse mitochondrial proteins. After database search, 29 acetylated sites in 26 proteins were identi?ed.     

Surface Activation of Poly(Methyl methacrylate) by Plasma Treatment: Stable Antibody Immobilization for Microfluidic Enzyme-Linked Immunosorbent Assay

With the aim of obtaining stable antibody immobilization on the poly(methyl methacrylate), PMMA channel surface, PMMA substrates were activated with O₂ plasma treatment to introduce surface polar groups on it. The plasma-treated PMMA surfaces were characterized using water contact angle measurement, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). It was observed that plasma treatment significantly improved the surface wettability with changing surface chemistry and topography. The strategy of immobilization of a model antibody, anti-goat IgG on plasma-treated PMMA involved two steps. First the plasma-treated PMMA was functionalized with (3-aminopropyl)thriethoxy silane, APTES off-chip which facilitated covalent capturing of antibody via a crosslinking agent in the inner surface of PMMA channel in the second step. The antibody immobilization on plasma-treated PMMA was also confirmed using AFM, XPS, and fluorescence microscopy. The anti-IgG covalently captured on channel surface was evaluated with sandwich ELISA protocol on-chip using fluorescence microscopy. The observed results demonstrate that this technique could be extended to integrate the current diagnostic techniques into the plastic chip for important biomarker diagnosis.     

A Multistep Enzyme Sensor for Sucrose Based on S-Layer Microparticles As Immobilization Matrix

Abstract

In this paper we report on the construction principle and performance of an amperometric 3-enzyme sensor for sucrose based on crystalline bacterial cell surface layers (S-layers) as immobilization matrix for the biological components.

Isoporous, crystalline surface layers (S-layers) have been identified as outermost cell envelope layer in many bacteria. Since they are composed of identical protein or glycoprotein subunits with functional groups in well defined positions and orientations, they represent ideal matrices for the controlled and reproducible immobilization of functional macromolecules, as required for the development of biosensors. Apart from single enzyme sensors, which were described earlier, a strikingly simple method for the assembly and optimization of multistep systems was developed. For the fabrication of an amperometric sucrose sensor invertase, mutarotase and glucose oxidase were individually immobilized on S-layer fragments isolated from Clostridium thermohydrosulfuricum L111-69 via aspartic acid as spacer molecules. Subsequently, appropriate mixtures of enzyme loaded S-layer fragments were deposited on a microfiltration membrane and finally, the composite multifunctional sensing layer was sputtered with gold in order to establish a good metal contact. Amperometric sucrose measurements based on H₂O₂ oxidation revealed a high signal level (1 μA^?1/cm^2?mmol sucrose), 5 min response time and a linear range up to 30 mM sucrose as the main characteristics of the S-layer sucrose sensor.