Thermoanalytical study of acid-treated clay containing amino acid immobilized on its surface

The aim of this study is to investigate the incorporation of amino acid molecules in an acid-activated montmorillonite by means of solid characterization after the incorporation of these biomolecules. The acid activation procedure was carried out for the purpose of increasing the acid sites in the clay as well as the impurity elimination in the mineral. Cysteine, aspartic, and glutamic acids were adsorbed on montmorillonite K10 which was previously treated with a hydrochloric acid solution. The clay was put in contact with amino acid solutions at two different concentrations. Each amino acid was adsorbed at identical conditions, with the pH fixed to ensure the charge of molecules and surface clay. The solid was characterized by means of X-ray diffraction, infrared spectroscopy, thermogravimetric analysis, and nitrogen adsorption at 77 K. After the amino acid adsorption, the powders showed changes in their characteristics as well as in their thermal behavior, which depended on both the concentration and the nature of the adsorbed amino acid. The thermal decomposition and elimination of cysteine occurred at a higher temperature than the aspartic and glutamic acid; the complete removal of glutamic acid molecules was not observed at 850 °C. The differences observed in the solid characteristic after the adsorption of each amino acid were discussed. Both the thermoanalytical study and characterization of materials after the interaction with amino acid molecules can be useful to understand the adsorption mechanism of biomolecules on solid surfaces.     

Conversion of Waste Tyres into Carbon Black and their Utilization as Adsorbent

Pyrolysis has the potential of transforming used tyres into useful recyclable products. Pyrolytic carbon black is one of the most important products of tyre pyrolysis. Waste tyres were pyrolysed at 450 °C in a batch reactor under atmospheric pressure. The recovered pyrolytic carbon black residues were studied to investigate their characteristics for use as a possible adsorbent. EDX elemental analysis and surface area determinations were used to investigate the distinctive features of pyrolytic carbon black. Due to various inorganic additives of the original tyre that contaminated the carbon black obtained, it was treated with acid for demineralization. The demineralized carbon black was activated at 900 °C in a furnace. It was observed that acid treatment and activation increased the surface areas and decreased the concentration of contaminants. Furthermore, adsorption characteristics of methylene blue on acid‐treated and activated carbon black (prepared via acid treatment) were compared with those of commercial activated carbon in liquid phase adsorption. It was found that adsorption capacity of methylene blue on acid‐treated activated carbon black was greater.     

Covalent Attachment of Bacteriorhodopsin Monolayer to Bromo‐terminated Solid Supports: Preparation,Characterization, and Protein Stability

The interfacing of functional proteins with solid supports and the study of related protein‐adsorption behavior are promising and important for potential device applications. In this study, we describe the preparation of bacteriorhodopsin (bR) monolayers on Br‐terminated solid supports through covalent attachment. The bonding, by chemical reaction of the exposed free amine groups of bR with the pendant Br group of the chemically modified solid surface, was confirmed both by negative AFM results obtained when acetylated bR (instead of native bR) was used as a control and by weak bands observed at around 1610 cm^?1 in the FTIR spectrum. The coverage of the resultant bR monolayer was significantly increased by changing the pH of the purple‐membrane suspension from 9.2 to 6.8. Although bR, which is an exceptionally stable protein, showed a pronounced loss of its photoactivity in these bR monolayers, it retained full photoactivity after covalent binding to Br‐terminated alkyls in solution. Several characterization methods, including atomic force microscopy (AFM), contact potential difference (CPD) measurements, and UV/Vis and Fourier transform infrared (FTIR) spectroscopy, verified that these bR monolayers behaved significantly different from native bR. Current–voltage (I–V) measurements (and optical absorption spectroscopy) suggest that the retinal chromophore is probably still present in the protein, whereas the UV/Vis spectrum suggests that it lacks the characteristic covalent protonated Schiff base linkage. This finding sheds light on the unique interactions of biomolecules with solid surfaces and may be significant for the design of protein‐containing device structures.     

Facile preparation of a high‐capacity boronate‐affinity adsorbent based on low‐cost commercial supports for selective enrichment of cis‐diol‐containing biomolecules

Boronate‐affinity adsorbents have been regarded as favorable extraction adsorbents for the pretreatment of cis‐diol‐containing biomolecules owning to their specific selectivity, but most of them have low adsorption capacity and a tedious synthesis methods. In this study, a new boronate‐affinity material (PGMA@FPBA) with high adsorption capacity was synthesized via a “one‐pot” method based on a low‐cost commercial support. The PGMA@FPBA was characterized by Fourier transform infrared spectroscopy (FT‐IR), X‐ray photoelectron spectroscopy (XPS), scanning electron microscope (SEM), and nitrogen adsorption/desorption measurements. The as‐prepared adsorbent showed good selectivity, high adsorption capacity (448 μmol/g for catechol), and fast adsorption equilibration (1 min) for cis‐diol‐containing biomolecules. Subsequently, as an example for application, the obtained PGMA@FPBA was used as a dispersive solid‐phase extraction (d‐SPE) adsorbent for enrichment of quercetin in red wine. The results indicated that the facile‐prepared boronate‐affinity adsorbent has great potential application for separation and enrichment of cis‐diol‐containing biomolecules in complex samples.     

Automated coating procedures to produce poly(ethylene glycol) brushes in fused‐silica capillaries

Many bioanalytical methods rely on electrophoretic separation of structurally labile and surface active biomolecules such as proteins and peptides. Often poor separation efficiency is due to surface adsorption processes leading to protein denaturation and surface fouling in the separation channel. Flexible and reliable approaches for preventing unwanted protein adsorption in separation science are thus in high demand. We therefore present new coating approaches based on an automated in‐capillary surface‐initiated atom transfer radical polymerization process (covalent coating) as well as by electrostatically adsorbing a presynthesized polymer leading to functionalized molecular brushes. The electroosmotic flow was measured following each step of the covalent coating procedure providing a detailed characterization and quality control. Both approaches resulted in good fouling resistance against the four model proteins cytochrome c, myoglobin, ovalbumin, and human serum albumin in the pH range 3.4−8.4. Further, even samples containing 10% v/v plasma derived from human blood did not show signs of adsorbing to the coated capillaries. The covalent as well as the electrostatically adsorbed coating were both found to be stable and provided almost complete suppression of the electroosmotic flow in the pH range 3.4−8.4. The coating procedures may easily be integrated in fully automated capillary electrophoresis methodologies.     

Chemical modification of natural clays

Natural clay samples and calcined clay varieties treated with hydrochloric acid of various strengths were characterized by X-ray diffraction, positron annihilation spectroscopy, and IR spectroscopy. The specific surfaces and pore sizes were determined by nitrogen adsorption. The constituent phases of the clay were found to be nontronite, muscovite, quartz, and hematite; after calcination, quartz, muscovite, hematite, and an amorphous phase remained in the samples. The Brilliant Green adsorption properties and tribotechnical properties of the modified clays were studied. The natural clay was surface-modified chemically by means of consecutive exposure to 36.5% hydrochloric acid and an alkaline hydrolyzate of rice hulls. Calcination at 700°С destroyed the amorphous phase to a greater extent; exposure to acid provided a greater recovery of aluminum and iron ions from the surface of calcined samples. Native clay composites with an alkaline hydrolyzate of rice hulls were shown to have enhanced tribotechnical characteristics.     

The role of ligands on protein retention in adsorption chromatography: A surface energetics approach

Protein adsorption onto hydrophobic chromatographic supports has been investigated using a colloid theory surface energetics approach. The surface properties of commercially available chromatographic beads, Toyopearl Phenyl 650‐C, and Toyopearl Butyl 650‐C, have been experimentally determined by contact angle and zeta potential measurements. The adsorption characteristics of these beads, which bear the same backbone matrix but harbor different ligands, have been studied toward selected model proteins, in the hydrated as well as dehydrated state. There were two prominent groups of proteins observed with respect to the chromatographic supports presented in this work: loosely retained proteins, which were expected to have lower average interaction energies, and the strongly retained proteins, which were expected to have higher average interaction energies. Results were also compared and contrasted with calculations derived from adsorbent surface energies determined by inverse liquid chromatography. These results showed a good qualitative agreement, and the interaction energy minima obtained from these extended Derjaguin, Landau, Verwey and Overbeek calculations were shown to correlate well with the experimentally determined adsorption behavior of each protein.     

Effect of dialysis treatment on the aggregation state of montmorillonite clay

In this work dialysis was used as a technique to modify the aggregation state of montmorillonite clay. Na-montmorillonite clay suspension was treated by a dialysis process and the effect of dialysis time on the surface area of the treated clay was studied. In a clay suspension, a decrease in cation content increases the interparticle repulsion forces exponentially, leading to separation of clay into the elementary clusters of layers by osmotic swelling. During dialysis treatment, the classical delamination process and mechanism were not observed. According to N(2) adsorption isotherms, the increase of surface area concerns to the development of mesoporosity. Nevertheless, the surface area increase cannot be explained by the delamination or exfoliation of some layers, since an increase in the expected number of layers that would yield the surface area does not explain the mesoporosity observed after dialysis. Based on these results, a new mechanism of evolution of the clay aggregation state after dialysis and drying is proposed.     

Removal of Disperse Dyes from Water Using Surfactant Treated Alumina

An alumina surface was modified by adsorption of an anionic surfactant, sodium dodecyl sulfate (SDS). Typical S‐shaped isotherm of surfactant on alumina was observed. The adsorption of Disperse Red‐11, Disperse Blue‐26 and Disperse Red‐156 on alumina and surfactant treated alumina has been investigated. The enhancement in adsorption of these disperse dyes on surfactant treated alumina is observed, which may be attributed to their solubilization in surfactant aggregates formed at the solid/liquid interface. The effect of pH on adsorption has been studied. The adsorption is greatly influenced by pH of the medium. The applicability of the Langmuir model and the Dual‐Mode sorption model (DSM) were tested for equilibrium data.     

Experimental study of albumin and lysozyme adsorption onto acrylic acid (AA) and 2-hydroxyethyl methacrylate (HEMA) surfaces

Many commercial soft contact lenses are based on poly-2-hydroxyethyl methacrylate (HEMA) and acrylic acid (AA) hydrogels. The adsorption of proteins, albumin and lysozyme, on such contact lens surfaces may cause problems in their applications. In this work the adsorption of proteins, albumin and lysozyme, on hydrogel surfaces, AA and HEMA, was investigated as a function of concentration of protein. Also the effects of pH and ionic strength of protein solution on the adsorption of protein were examined. The obtained results indicated that the degree of adsorption of protein increased with the concentration of protein, and the adsorption of albumin on HEMA surface at the studied pHs (6.2-8.6) was higher than AA surface, whereas the adsorption of lysozyme on AA surface at the same pHs was higher than HEMA. The change in ionic strength of protein solution affected the proteins adsorption on both AA and HEMA surfaces. Also, the amount of sodium ions deposited on the AA surface was much higher than HEMA surface. This effect can be related to the negative surface charge of AA and its higher tendency for adsorption of sodium ions compared to the HEMA surface.     

Understanding the Adsorption of Peptides and Proteins onto PEGylated Gold Nanoparticles

Polyethylene glycol (PEG) surface conjugations are widely employed to render passivating properties to nanoparticles in biological applications. The benefits of surface passivation by PEG are reduced protein adsorption, diminished non-specific interactions, and improvement in pharmacokinetics. However, the limitations of PEG passivation remain an active area of research, and recent examples from the literature demonstrate how PEG passivation can fail. Here, we study the adsorption amount of biomolecules to PEGylated gold nanoparticles (AuNPs), focusing on how different protein properties influence binding. The AuNPs are PEGylated with three different sizes of conjugated PEG chains, and we examine interactions with proteins of different sizes, charges, and surface cysteine content. The experiments are carried out in vitro at physiologically relevant timescales to obtain the adsorption amounts and rates of each biomolecule on AuNP-PEGs of varying compositions. Our findings are relevant in understanding how protein size and the surface cysteine content affect binding, and our work reveals that cysteine residues can dramatically increase adsorption rates on PEGylated AuNPs. Moreover, shorter chain PEG molecules passivate the AuNP surface more effectively against all protein types.     

Investigation of the Antifouling Properties of Phenyl Phosphorylcholine‐Based Modified Gold Surfaces

Low impedance, antifouling coatings on gold electrodes based on three new zwitterionic phenyl phosphorylcholine (PPC)‐based layers namely 1) reductively adsorbed PPC diazonium salt, 2) dithiocarbamate PPC SAM and 3) lipoamide PPC SAM (PPC coupled to α‐lipoic acid) were evaluated. The layers were assessed for their ability to limit nonspecific adsorption of proteins to electrode surface with some significant differences observed compared with previously studied PPC diazonium salts reductively adsorbed on glassy carbon. Fluorescence microscopy and electrochemical impedance spectroscopy results suggest that protein adsorption is sensitive to the difference in the structure of the PPC molecules and the charge neutrality of the layers. The lipoamide PPC SAM was shown to be the most effective at resisting nonspecific protein adsorption and this layer was as effective as the ‘gold standard’ of oligo(ethylene oxide) SAMs on gold and PPC diazonium salts reductively adsorbed on glassy carbon.     

Facile immobilization of biomolecules onto various surfaces using epoxide-containing antibiofouling polymers

The surface modifications of plastic or glass substrate and the subsequent immobilization of biomolecules onto the surfaces has been a central feature of the fabrication of biochips. To this end, we designed and synthesized new epoxide-containing random copolymers that form stable polymer adlayers on plastic or glass surface and subsequently react with amine or sulfhydryl functional groups of biomolecules under aqueous conditions. Epoxide-containing random copolymers were synthesized by radical polymerization of three functional monomers: a monomer acting as an anchor to the surfaces, a PEG group for preventing nonspecific protein adsorption, and an epoxide group for conjugating to biomolecules. Polymer coating layers were facilely formed on cyclic olefin copolymer (COC) or glass substrate by simply dipping each substrate into a solution of each copolymer. The polymer-coated surfaces characterized by a contact angle analyzer and X-ray photoelectron spectroscopy (XPS) showed very low levels of nonspecific immunoglobulin G (IgG) adsorption compared to the uncoated bare surface (control). Using a microcontact printing (μCP) method, antibodies as representative biomolecules could be selectively attached onto the copolymers-coated glass or COC surface with high signal-to-noise ratios.     

粘土矿物-蛋白质超薄膜的设计与生物催化性能

随着生物芯片及生物大分子有序自组装技术的发展,蛋白质-粘土矿物的超薄复合膜（clay-protein ultrathin films,CPUFs）的制备在生物催化领域引起人们的广泛关注.本文详述了蛋白质（溶解酵素、牛血清白蛋白、木瓜蛋白酶与精蛋白）与单片粘土（elementary sheet）矿物（钠化皂石）形成单层或多层纳米薄膜复合物的吸附过程及二维分子自组装的制备工艺,对交替层吸附（layer-by-layer,LbL）和Langmuir-Blodgett（LB）方法应用于CPUFs的构筑分别进行了介绍,并阐述了紫外可见分光光度计（UV-vis）,衰减全反射红外光谱（ATR-FTIR）,X射线衍射（XRD）,原子力显微镜（AFM）和界面化学技术等手段对薄膜定性定量表征的研究方法及结果.研究表明,在CPUFs的形成过程中,静电相互作用是一个突出因素,但不是唯一驱动力.在利用LB技术构筑CPUFs的研究中,我们发现即使在无表面活性剂分子的协助下,水溶性蛋白质也能够在粘土矿物稀溶液的界面上形成较为稳定的蛋白质-粘土矿物Langmuir复合膜.通过研究表面压力与时间（？-t）的动力学曲线和表面压力对面积（？-A）的等温线,实现了粘土矿物与蛋白分子吸附过程即CPUFs形成过程的实时监测,并测定出CPUFs中蛋白质含量（NS）、蛋白质分子堆积密度（？？）、单个蛋白质分子在粘土片层上的平均占位面积（？？）及蛋白质在粘土层的饱和吸附量等物理化学参数.通过将界面化学测定结果、光谱学方法测定结果与粘土矿物体相溶液吸附实验结果相比较,发现LB技术实现了单层粘土片（厚度约1.3nm）的吸附与自组装,证明界面化学方法是定量化研究CPUFs的一种有效手段.通过将含有溶解酵素的CPUFs膜用于流水池反应器上进行生物活性检测,考察了其在睾丸酮丛毛单胞菌生长发育过程中的催化性能,结果表明LZ分子被锚固在CPUFs上以后,未发生明显的生物活性失活现象.     

Synthesis of large‐pore mesostructured cellular foam silica spheres for the adsorption of biomolecules

Mesostructured cellular foam (MCF) silica spheres with different textual parameters were synthesized using a triblock copolymer as a template. The effects of acid concentration and aging time on the window size and morphology were discussed. Besides, the adsorption performances of lysozyme and bovine serum albumin on the blank MCF with different window size and aminopropyl‐modified MCFs were studied. The adsorption capacity and rates were shown to be dependent on the window size and surface chemical properties of the adsorbents. In particular, the MCF with window size of 15.2 nm showed fast adsorption for lysozyme with an adsorption capacity of 500 mg/g in 10 min. Furthermore, it has been shown that MCF spheres are potential materials in the separation of biomolecules because of their chemical tunable surface and molecular sieve properties.     

Clay-protein ultrathin films: Design and bio-catalytic performance study

In recent years,immense interest has been paid to the biomolecular architecture with the aim of protein assembly in two dimensions on solid substrates,and the constructions of clay-protein ultrathin films(CPUFs) are particularly concerned.This paper gives an overview of the recent research concerning the protein molecules(lysozyme,papain,protamine,bovine serum albumin) immobilized on clay mineral(Na-saponite) platelets and assembled in monolayered or multilayered hybrid ultrafilms or nanofilms.Two techniques including alternate layer-by-layer(LbL) assembly and the Langmuir-Blodgett(LB) are described in detail.A variety of means,including UV-vis absorption,attenuated total reflectance Fourier transform infrared(ATR-FTIR) spectroscopy,XRD,AFM and surface chemistry techniques,have been described for characterization of the films in terms of quantification of protein and clay.The result reveals that electrostatic interaction is a prominent but not the only driving force in CPUF construction.In the case of LB technique,we managed to manipulate the elementary clay mineral platelets(1.3 nm in thickness) and assemble proteins into CPUFs with the aid of surfactants,and the formation of CPUFs was monitored via surface pressure vs.time(π-t) kinetics curves and surface pressure vs.area(π-A) isotherms.The factors that influence protein adsorption on the clay layer,such as surfactants,the concentration of clay,equilibrium time,categories of protein,and injection methods,were investigated.The parameters such as protein amount(nS),packing density(■),and average surface area per molecule(Ω) of deposited CPUFs were measured via method of surface chemistry and spectroscopy.By comparing the results of surface chemistry with those of adsorption experiments,we demonstrate that the surface chemistry method is a useful tool in investigating CPUFs.We also found that the water soluble protein molecules could form protein-clay hybrid monolayer over the dilute clay dispersions without addition of surfactants,and CPUFs containing elementary clay sheets and protein with great homogeneity were easily prepared by controlling certain surface pressure.To investigate the bio-catalytic performance of the immobilized lysozyme in CPUFs,we deposited CPUFs onto a cover glass,and installed the cover glass in a flow cell-grown reactor for Comamonas testosteroni(WDL7-GFP) incubation.The results show that the proliferation of WDL7-GFP is greatly suppressed by lysozyme,which demonstrates that lysozyme still retains its bioactivity after it is immobilized in the CPUFs.     

Profiling protein-surface interactions of multicomponent suspensions via flow cytometry

This study presents the use of flow cytometry as a high-throughput quantifiable technique to study multicomponent adsorption interactions between proteins and surfaces. Flow cytometry offers the advantage of high-throughput analysis of multiple parameters on a very small sampling scale. This enables flow cytometry to distinguish between individual adsorbent particles and adsorbate components within a suspension. As a proof of concept study, the adsorption of three proteins--bovine serum albumin (BSA), bovine immunoglobulin gamma (IgG) and fibrinogen--onto five surface-modified organosilica microsphere surfaces was used as a model multicomponent system for analysis. By uniquely labeling each protein and solid support type with spectrally distinguishable fluorescent dyes, the adsorption process could be "multiplexed" allowing for simultaneous screening of multiple adsorbate (protein) and adsorbent (particle surface) interactions. Protein adsorption experiments quantified by flow cytometry were found to be comparable to single-component adsorption studies by solution depletion. Quantitative distribution of the simultaneous competitive adsorption of BSA and IgG indicated that, at concentrations below surface saturation, both proteins adsorbed onto the surface. However, at concentrations greater than surface saturation, BSA preferentially adsorbed. Multiplexed particle suspensions of optically encoded particles were modified to produce a positively and negatively charged surface, a grafted 3400 MW poly(ethylene glycol) layer, or a physisorbed BSA or IgG layer. It was observed that adsorption was rapid and irreversible on all of the surfaces, and preadsorbed protein layers were the most effective in preventing further protein adsorption.     

Immobilization of metallothionein on highly oriented pyrolytic graphite for biosensor design

Immobilization of protein molecules on solid supports or surfaces in a controlled fashion is an important task for protein analysis at the solid/solution or solid/gas interface and biosensor fabrication. In this paper, the structure and biological activities of metallothionein (MT) layers immobilized on highly oriented pyrolytic graphite (HOPG) surfaces by means of two different strategies based on unspecific adsorption/chemisorption (MT‐HOPG system) and covalent binding (MT‐modified HOPG system) were studied respectively. The MT layers obtained by covalent binding to a previously functionalized HOPG surface are smooth and show a close‐packed ordered monolayer in contrast to those obtained by direct adsorption of the protein on substrate, which are disordered and relatively rough. Both adsorbed proteins exhibit reversible electron transfer at 0.25 V (Ag/AgCl) after immersion in CuSO₄ solution, whereas redox current of MT‐modified HOPG system is four times larger than that of MT‐HOPG system. Moreover, the MTs adsorbed on bare HOPG surfaces are obviously denatured. All the above results show that covalent binding strategies lead to high structural regularity and mechanical stability of the adsorbed protein molecules with a maintained biological activity, which is prospective for applications in immobilizing MT on a transducer for biosensor design. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of exchanged surfactant cations on the pore structure and adsorption characteristics of montmorillonite

Ca-montmorillonite (Ca-Mont) was exchanged with two quaternary amines, tetramethylammonium (TMA) chloride and hexadecyltrimethylammonium (HDTMA) bromide, to study the surfactant ion exchange effect on the pore structure, surface characteristics, and adsorption properties of montmorillonite. The revolution of both the surface area and pore structure of montmorillonite was characterized based on classical and fractal analyses of the nitrogen isotherms as well as the XRD patterns. The change of surface characteristics was identified from FTIR patterns and zeta-potential plots. The adsorption isotherms of acid dye, Amido Naphthol Red G (AR1), were then measured to identify the effects of the ion-exchange process on the adsorption properties of montmorillonite. It was found that the exchange processes might induce an increase or decrease in the surface area, pore size, pore volume, and surface fractal dimension D of montmorillonite, depending on the size, the molecular arrangement, and the degree of hydration of the exchanged ion in the clay. On the other hand, it was also found that the hydrophobic bonding by conglomeration of large C(16) alkyl groups associated with HDTMA could cause positive charge development on the surface of montmorillonite, which was not observed for TMA-modified montmorillonite (TMM). The effects of the alteration of the surface characteristics of montmorillonites on their adsorption selectivity for acid dye were discussed.     

Oriented attachment and membrane reconstitution of His-tagged cytochrome c oxidase to a gold electrode: in situ monitoring by surface-enhanced infrared absorption spectroscopy

A novel concept is introduced for the oriented incorporation of membrane proteins into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent was immobilized on a chemically modified gold surface via the affinity of its histidine-tag to a nickel-chelating nitrilo-triacetic acid (NTA) surface. The oriented protein monolayer was reconstituted into the lipid environment by detergent substitution. The individual steps of the surface modification, including (1) chemical modification of the gold support, (2) adsorption of the protein, and (3) reconstitution of the lipid bilayer, were followed in situ by means of surface-enhanced infrared absorption spectroscopy (SEIRAS) and accompanied by normal-mode analysis. The high surface sensitivity of SEIRAS allows for the identification of each chemical reaction process within the monolayer at the molecular level. Finally, full functionality of the surface-tethered cytochrome c oxidase was demonstrated by cyclic voltammetry after binding of the natural electron donor cytochrome c.