Isolation and pre-concentration of basic proteins in aqueous mixture via solid-phase extraction with multi-walled carbon nanotubes assembled on a silica surface

Multi-walled carbon nanotubes (MWNTs) were assembled on a silica surface using a polyelectrolyte-assisted layer-by-layer (LBL) assembly technique. The surface-assembled silica spheres with MWNTs (MWNTs/SiO(2)), which serve as a novel solid-phase extraction sorbent for separation/pre-concentration of basic proteins, was investigated. The adsorption behavior of cytochrome c (cyto-c) by MWNTs/SiO(2) spheres agrees well with the Langmuir adsorption model. A thorough scrutiny of the experimental parameters affecting the adsorption of cyto-c from aqueous solution onto the MWNTs/SiO(2) spheres and its subsequent desorption was carried out. A maximum adsorption capacity of cyto-c was derived as 112 mg (cyto-c) g(-1) (MWNTs). A distinct feature of the MWNTs/SiO(2)-packed micro-column provides clear advantages of minimized flow impedance when operated in a flow system, in addition to better separation efficiency as well as the favorable enrichment capability of proteins, characterized by an enrichment factor of 30 by using 2.0 mL of aqueous solution. The practical applicability of the MWNTs/SiO(2) spheres as a sorbent for the isolation of basic proteins from acidic protein species was demonstrated by effective separation of cyto-c from bovine serum albumin (BSA).     

Preparation of Keggin‐type phosphomolybdate by a one‐step solid‐state reaction at room temperature and its application in protein adsorption

Keggin‐type phosphomolybdate ((C₁₉H₄₂N)₃PMo₁₂O₄₀) is prepared by a one‐step solid‐state reaction at room temperature and characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, X‐ray diffraction, thermogravimetric analysis, and elemental analysis. The as‐prepared phosphomolybdate is demonstrated to be an efficient adsorbent for proteins. In this particular case, the selective adsorption of neutral protein hemoglobin is achieved. While under the same conditions virtually no adsorption of acidic and basic proteins, represented by bovine serum albumin and cytochrome c, are observed. A solid‐phase extraction procedure is developed for the selective isolation of hemoglobin. At pH 6, a sorption efficiency of 91.4% is achieved for 100 μg/mL hemoglobin in 1.0 mL solution by using 5.0 mg of the phosphomolybdate. The adsorption behavior of hemoglobin fits well with a Langmuir adsorption model, corresponding to a theoretical adsorption capacity of 55.86 mg/g. The retained hemoglobin could be readily recovered by using a 60 mmol/L imidazole solution at pH 7, giving rise to a recovery of 64.7%. The practical application of phosphomolybdate for protein adsorption is demonstrated by the selective isolation of hemoglobin from human whole blood followed by a sodium dodecyl sulfate polyacrylamide gel electrophoresis assay.     

Multifunctional nanoparticles composite for MALDI-MS: Cd2+-doped carbon nanotubes with CdS nanoparticles as the matrix, preconcentrating and accelerating probes of microwave enzymatic digestion of peptides and proteins for direct MALDI-MS analysis

For the first time, we utilized multifunctional nanoparticles composite (NPs composite) for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis of peptides and proteins. Multiwalled carbon nanotubes doped with Cd(2+) ions and modified with cadmium sulfide NPs were synthesized by a chemical reduction method at room temperature. The multifunctional NPs composite applied for the analysis of peptides and microwave-digested proteins in the atmospheric pressure matrix-assisted laser desorption/ionization ion-trap and MALDI time-of-flight (TOF) mass spectrometry (MS) was successfully demonstrated. The maximum detection sensitivity for peptides in MALDI-MS was achieved by the adsorption of negatively charged peptides onto the surfaces of NP composite through electrostatic interactions. The optimal conditions of peptide mixtures were obtained at 20 min of incubation time using 1 mg of NPs composite when the pH of the sample solution was kept higher than the pI values of peptides. The potentiality of the NP composite in the preconcentration of peptides was compared with that of the individual NP by calculating the preconcentration factors (PF) and found that the NPs composite showed a 4-6 times of PF than the other NPs. In addition, the NPs composite was also applied as heat-absorbing materials for efficient microwave tryptic digestion of cytochrome c and lysozyme from milk protein in MALDI-TOF-MS analysis. We believe that the use of NPs composite technique would be an efficient and powerful preconcentrating tool for MALDI-MS for the study of proteome research.     

固定化铜离子亲和膜色谱柱吸附血红蛋白的研究

将纤维素滤纸进行碱处理及环氧活化、偶联亚氨基二乙酸、固定化铜离子等处理,并将其装入自制的色谱柱管,制得固定化铜离子亲和膜色谱柱。该柱可用于吸附血红蛋白(hemoglobin,Hb),吸附率可达到90%以上。考察了上样量、pH值、温度、上样速度等因素对固定化铜离子亲和膜吸附Hb的影响。实验结果表明,固定化铜离子亲和膜色谱柱吸附血红蛋白的最佳条件为：室温下实验,缓冲体系的pH值控制在6~8,上样速度0.5~1.0 mL/min,上样量为3.16~7.90 mg/g。     

水杨酸型螯合吸附材料对重金属离子的吸附性能

将5-氨基水杨酸接枝到PGMA/SiO2微粒的聚甲基丙烯酸缩水甘油酯(PGMA)大分子链上,成功制备了一种新型螯合吸附材料ASA-PGMA/SiO2。采用静态法研究了ASA-PGMA/SiO2对重金属离子Cu2+、Cd2+、Zn2+、Pb2+的吸附性能,结果表明其对Cu2+、Cd2+、Zn2+、Pb2+具有很强的螯合吸附能力,吸附容量分别可以达到0.42、0.40、0.35、0.31mmol/g。体系的pH对吸附容量影响较大,吸附行为服从Langmuir和Freundlich吸附模型。使用0.1mol/L的盐酸溶液就可实现重金属离子的解吸。通过反复吸附-解吸实验证明ASA-PGMA/SiO2具有良好的重复使用性能。     

Poly(amidoxime)-reduced graphene oxide composites as adsorbents for the enrichment of uranium from seawater

The development of efficient materials for high extraction of uranium(UO22+) from seawater is critical for nuclear energy. Poly(amidoxime)-reduced graphene oxide(PAO/rGO) composites with excellent adsorption capability for UO22+ were synthesized by in situ polymerization of acrylonitrile monomers on GO surfaces, followed by amidoximation treatment with hydroxylamine. The adsorption capacities of PAO/rGO composites for UO22+ reached as high as 872 mg/g at pH 4.0. The excellent tolerance of these composites for high salinity and their regeneration-reuse properties can be applied in the nuclear-fuel industry by high extraction of trace UO22+ ions from seawater.     

Removal of Mn(II) from Acidic Wastewaters Using Graphene Oxide–ZnO Nanocomposites

Pollution due to acidic and metal-enriched waters affects the quality of surface and groundwater resources, limiting their uses for various purposes. Particularly, manganese pollution has attracted attention due to its impact on human health and its negative effects on ecosystems. Applications of nanomaterials such as graphene oxide (GO) have emerged as potential candidates for removing complex contaminants. In this study, we present the preliminary results of the removal of Mn(II) ions from acidic waters by using GO functionalized with zinc oxide nanoparticles (ZnO). Batch adsorption experiments were performed under two different acidity conditions (pH1 = 5.0 and pH2 = 4.0), in order to evaluate the impact of acid pH on the adsorption capacity. We observed that the adsorption of Mn(II) was independent of the pH_PZC value of the nanoadsorbents. The qmax with GO/ZnO nanocomposites was 5.6 mg/g (34.1% removal) at pH = 5.0, while with more acidic conditions (pH = 4.0) it reached 12.6 mg/g (61.2% removal). In turn, the results show that GO/ZnO nanocomposites were more efficient to remove Mn(II) compared with non-functionalized GO under the pH2 condition (pH2 = 4.0). Both Langmuir and Freundlich models fit well with the adsorption process, suggesting that both mechanisms are involved in the removal of Mn(II) with GO and GO/ZnO nanocomposites. Furthermore, adsorption isotherms were efficiently modeled with the pseudo-second-order kinetic model. These results indicate that the removal of Mn(II) by GO/ZnO is strongly influenced by the pH of the solution, and the decoration with ZnO significantly increases the adsorption capacity of Mn(II) ions. These findings can provide valuable information for optimizing the design and configuration of wastewater treatment technologies based on GO nanomaterials for the removal of Mn(II) from natural and industrial waters.     

Magnetic dye affinity beads for the adsorption of beta-casein

Casein is well known as a good protein emulsifier and beta-casein is the major component of casein and commercial sodium caseinate. Dye affinity adsorption is increasingly used for protein separation. beta-Casein adsorption onto Reactive Red 120 attached magnetic poly(2-hydroxyethyl methacrylate) (m-PHEMA) beads was investigated in this work. m-PHEMA beads (80-120 microm in diameter) were produced by dispersion polymerization. The dichlorotriazine dye Reactive Red 120 was attached covalently as a ligand. The dye attached beads, having a swelling ratio of 55% (w/w) and carrying different amounts of Reactive Red 120 (9.2 micromol . g(-1)-39.8 micromol . g(-1)), were used in beta-casein adsorption studies. The effects of the initial concentration, pH, ionic strength and temperature on the adsorption efficiency of dye attached beads were studied in a batch reactor. The non-specific adsorption on the m-PHEMA beads was 1.4 mg . g(-1). Reactive Red 120 attachment significantly increased the beta-casein adsorption up to 37.3 mg . g(-1). More than 95.4% of the adsorbed beta-casein was desorbed in 1 h in a desorption medium containing 1.0 M KSCN at pH 8.0. We concluded that Reactive Red 120 attached m-PHEMA beads can be applied for beta-casein adsorption without significant losses in the adsorption capacities.     

阴离子交换树脂固相萃取分离全血中血红蛋白的研究

以330阴离子交换树脂为吸附材料建立了固相萃取分离血红蛋白的方法。利用血红蛋白和树脂之间的疏水作用力将血红蛋白吸附到树脂上,以Tris—HCl缓冲液（PH-8．9）为洗脱剂回收血红蛋白。考察了溶液PH值、吸附时间、离子强度、洗脱剂的种类及其酸度等对分离纯化效率的影响。在最优实验条件下,树脂对血红蛋白的吸附率和洗脱率分别为87％和70％,吸附容量为42．9μg／mg。吸收光谱和SDS—PAGE凝胶电泳证明,该方法可有效地从人全血中分离出纯度较高的血红蛋白。     

Control of Enzyme-Solid Interactions via Chemical Modification

Electrostatic forces could contribute significantly toward enzyme-solid interactions, and controlling these charge-charge interactions while maintaining high affinity, benign adsorption of enzymes on solids is a challenge. Here, we demonstrate that chemical modification of the surface carboxyl groups of enzymes can be used to adjust the net charge of the enzyme and control binding affinities to solid surfaces. Negatively charged nanosolid, α-Zr(HPO(4))(2)·H(2)O (abbreviated as α-ZrP) and two negatively charged proteins, glucose oxidase (GO) and methemoglobin (Hb), have been chosen as model systems. A limited number of the aspartate and glutamate side chains of these proteins are covalently modified with tetraethylenepentamine (TEPA) to convert these negatively charged proteins into the corresponding positively charged ones (cationized). Cationized proteins retained their structure and activities to a significant extent, and the influence of cationization on binding affinities has been tested. Cationized GO, for example, showed 250-fold increase in affinity for the negatively charged α-ZrP, when compared to that of the unmodified GO, and cationized Hb, similarly, indicated 26-fold increase in affinity. Circular dichroism spectra showed that α-ZrP-bound cationized GO retained native-like structure to a significant extent, and activity studies showed that cationized GO/α-ZrP complex is ～2.5-fold more active than GO/α-ZrP. Cationized Hb/α-ZrP retained ～75% of activity of Hb/α-ZrP. Therefore, enzyme cationization enhanced affinities by 1-2 orders of magnitude, while retaining considerable activity for the bound biocatalyst. This benign, chemical control over enzyme charge provided a powerful new strategy to rationally modulate enzyme-solid interactions while retaining their biocatalytic properties.     

SiO2/graphene composite for highly selective adsorption of Pb(II) ion

SiO(2)/graphene composite was prepared through a simple two-step reaction, including the preparation of SiO(2)/graphene oxide and the reduction of graphene oxide (GO). The composite was characterized by UV-Vis spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscope, and X-ray photoelectron spectroscopy, and what is more, the adsorption behavior of as-synthesized SiO(2)/graphene composite was investigated. It was interestingly found that the composite shows high efficiency and high selectivity toward Pb(II) ion. The maximum adsorption capacity of SiO(2)/graphene composite for Pb(II) ion was found to be 113.6 mg g(-1), which was much higher than that of bare SiO(2) nanoparticles. The results indicated that SiO(2)/graphene composite with high adsorption efficiency and fast adsorption equilibrium can be used as a practical adsorbent for Pb(II) ion.     

Fabrication of a novel hydrophobic/ion‐exchange mixed‐mode adsorbent for the dispersive solid‐phase extraction of chlorophenols from environmental water samples

A novel mixed‐mode adsorbent was prepared by functionalizing silica with tris(2‐aminoethyl)amine and 3‐phenoxybenzaldehyde as the main mixed‐mode scaffold due to the presence of the plentiful amino groups and benzene rings in their molecules. The adsorption mechanism was probed with acidic, natural and basic compounds, and the mixed hydrophobic and ion‐exchange interactions were found to be responsible for the adsorption of analytes. The suitability of dispersive solid‐phase extraction was demonstrated in the determination of chlorophenols in environmental water. Several parameters, including sample pH, desorption solvent, ionic strength, adsorbent dose, and extraction time were optimized. Under the optimal extraction conditions, the proposed dispersive solid‐phase extraction coupled with high‐performance liquid chromatography showed good linearity range and acceptable limits of detection (0.22∽0.54 ng/mL) for five chlorophenols. Notably, the higher extraction recoveries (88.7∽109.7%) for five chlorophenols were obtained with smaller adsorbent dose (10 mg) and shorter extraction time (15 min) compared with the reported methods. The proposed method might be potentially applied in the determination of trace chlorophenols in real water samples.     

Application of central composite design to the optimisation of aqueous two-phase extraction of human antibodies

The partition of human antibodies in aqueous two-phase systems (ATPSs) of polyethylene glycol (PEG) and phosphate was systematically studied using first pure proteins systems and then an artificial mixture of proteins containing 1mg/ml human immunoglobulin G (IgG), 10mg/ml serum albumin and 2mg/ml myoglobin. Preliminary results obtained using pure proteins systems indicated that the PEG molecular weight and concentration, the pH value and the salts concentration had a pronounced effect on the partitioning behaviour of all proteins. For high ionic strengths and pH values higher than the isoelectric point (pI) of the contaminant proteins, IgG could be selectively recovered on the top phase. According to these results, a face centred composite design was performed in order to optimise the purification of IgG from the mixture of proteins. The optimal conditions for the isolation of IgG were observed for high concentrations of NaCl and low concentrations of both phase forming components. The best purification was achieved using an ATPS containing 8% (w/w) PEG 3350, 10% (w/w) phosphate pH 6 and 15% (w/w) NaCl. A recovery yield of 101+/-7%, a purity of 99+/-0% and a yield of native IgG of 97+/-4% were obtained. Back extraction studies of IgG to a new phosphate phase were performed and higher yields were obtained using 10% phosphate buffer at pH 6. The total extraction yield was 76% and the purity 100%.     

Solid-phase extraction of antimony using chemically modified SiO2-PAN nanoparticles

A new analytical method using 1-(2-pyridylazo)-2-naphthol (PAN)-modified SiO2 nanoparticles as solid-phase extractant has been developed for the preconcentration of trace amounts of Sb(III) in different water samples. Conditions of the analysis such as preconcentration factor, effect of pH, sample volume, shaking time, elution conditions, and effects of interfering ions for the recovery of the analyte were investigated. The adsorption capacity of nanometer SiO2-PAN was found to be 186.25 micromol/g at optimum pH and the LOD (3sigma) was 0.60 microg/L. The extractant showed rapid kinetic sorption. The adsorption equilibrium of Sb(III) on nanometer SiO2-PAN was achieved in 10 min. Adsorbed Sb(III) was easily eluted with 4 mL 2 M hydrochloric acid. The maximum preconcentration factor was 62.20. The method was applied for the determination of trace amounts of Sb(III) in various water samples (tap, mineral water, and industrial effluents).     

Piezoelectric Quartz Crystal Anti‐Protein Immunosensors Based on Immobilized Fullerene C60‐Proteins

Various fullerene C60‐proteins such as C60‐myoglobin (C60‐Mb), C60‐hemoglobin (C60‐Hb) and C60‐gliadin, coated piezoelectric quartz crystals were prepared and applied in piezoelectric quartz crystal immunosensors for protein‐antibodies such as anti‐myoglobin (Anti‐Mb), anti‐hemoglobin (Anti‐Hb) and anti‐gliadin respectively. The immobilizations of myoglobin, hemoglobin and gliadin onto Fullerene C60 were studied with a C60‐coated piezoelectric crystal detection system, respectively. The partially irreversible frequency responses for theses proteins were observed by a desorption study, implying that C60 can strongly adsorb these proteins. Thus, immobilized C60‐Mb, C60‐Hb and C60‐gliadin coating materials were successfully prepared and identified with FTIR spectrometry. The C60‐Mb, C60‐Hb and C60‐gliadin coated piezoelectric (PZ) quartz crystal immunosensors with homemade computer interfaces for signal acquisition and data processing were developed and applied for detection of Anti‐Mb, Anti‐Hb and anti‐gliadin respectively. The C60‐protein coated PZ immunosensors for Anti‐Mb, Anti‐Hb and antigliadin exhibited linear frequency responses to the concentrations of theses anti‐proteins with sensitivities of 1.43 × 10³, 2.59 × 10³ and 8.05 × 10³ Hz/(mg/mL) respectively. The detection limits of these PZ‐immunosensors were 4.36 × 10^?3, 3.23 × 10^?3 and 1.98 × 10^?3 mg/mL for Anti‐Mb, Anti‐Hb and anti‐gliadin respectively. Effects of pH and temperature on the frequency responses of the anti‐protein PZ‐immunosensors were also investigated. The optimum pH of these anti‐proteins and the optimum temperature for the PZ‐immunosensors were observed at pH = 7 and around 30 °C respectively. The interferences of various common species in human blood, e.g., cysteine, tyrosine, urea, glucose, ascorbic acid and metal ions, to these anti‐protein PZ‐immunosensors were also investigated respectively. These species showed nearly no interference or quite small interference with the anti‐protein PZ‐immunosensors. The reproducibility and lifetime of these immobilized C60‐protein coated PZ crystal immunosensors were also investigated and discussed.     

In situ self‐assembled reduced graphene oxide aerogel embedded with nickel oxide nanoparticles for the high‐efficiency separation of ovalbumin

A three‐dimensional reduced graphene oxide aerogel with embedded nickel oxide nanoparticles was prepared by a one‐step self‐assembly reaction in a short time. The nanoparticles could be captured into the interior of reduced graphene oxide network during the formation of the three‐dimensional architecture. The composite exhibited porosity, good biocompatibility, and abundant metal affinity binding sites. The aerogel was used to isolate ovalbumin selectively from egg white, and favorable adsorption was achieved at pH 3. An adsorption efficiency of 90.6% was obtained by using 1 mg of the composite for adsorbing 70 μg/mL of ovalbumin in 1.0 mL of sample solution, and afterwards a recovery of 90.7% was achieved by using an eluent of 1.0 mL Britton–Robinson buffer solution at pH 5. After the adsorption/desorption, ovalbumin showed no change in the conformation. The adsorption behavior of ovalbumin on the reduced graphene oxide composite well fitted to the Langmuir adsorption model, and a corresponding theoretical maximum adsorption capacity was 1695.2 mg/g. A sodium dodecyl sulfate polyacrylamide gel electrophoresis assay demonstrated that the aerogel could selectively isolate ovalbumin from chicken egg white.     

Adsorption of Zn(II) on graphene oxide prepared from low‐purity of amorphous graphite

The adsorption of Zn(II) in aqueous solutions on graphene oxide (GO) prepared from low‐purity of natural amorphous graphite has been studied in this work. The study was performed through the measurements of Zeta potential, atomic force microscope, Fourier transform infrared spectrum and X‐ray photoelectron spectroscopy. The results indicated that the adsorption followed the Langmuir model with the maximum Zn(II) adsorption capacity of 73 mg/g at pH 7.0. In addition, the adsorption was well described by the pseudo‐second‐order kinetics model. The mechanism of the Zn(II) adsorption on GO was mainly attributed to chemical adsorption through complexation reaction between Zn(II) and hydroxyl or carboxyl groups on the GO sheets, while the electrostatic interaction also contribute to the whole interaction. Copyright © 2016 John Wiley & Sons, Ltd.     

The human heart proteome: Two-dimensional maps using narrow-range immobilised pH gradients

The analysis of complex proteomes is undertaken using a variety of techniques and technologies such as 2-DE, surface-enhanced laser desorption ionisation, and various types of MS. In order to overcome the complexities of protein expression in discrete proteomes, sample fractionation has become an important aspect of proteomic experiments. The use of narrow-range IPGs (nrIPGs) is of special importance using the 2-DE proteomics workflow, since an enhanced visualisation of a given proteome is achieved through an improved physical separation and resolution of proteins. The work described in this paper presents a series of protein maps of the human heart left ventricle proteome that have been generated using nrIPGs for the first, IEF, dimension of 2-DE. A total of 374 gel spots were excised from seven different pH gradients, covering the range pH 3-10, giving rise to a total of 388 identifications from 110 unique proteins. Using Gene Ontologies (GOs), the identified proteins were found to be associated with 97 types of GO Process, 144 types of GO Function, and 54 types of GO Component. It is hoped that the maps presented in this paper will be of use to other researchers for reference purposes.     

Characterizing the adsorption of proteins on glass capillary surfaces using electrospray-differential mobility analysis

We quantify the adsorption and desorption of a monoclonal immunoglobulin-G antibody, rituxamab (RmAb), on silica capillary surfaces using electrospray-differential mobility analysis (ES-DMA). We first develop a theory to calculate coverages and desorption rate constants from the ES-DMA data for proteins adsorbing on glass capillaries used to electrospray protein solutions. This model is then used to study the adsorption of RmAb on a bare silica capillary surface. A concentration-independent coverage of ≈4.0 mg/m(2) is found for RmAb concentrations ranging from 0.01 to 0.1 mg/mL. A study of RmAb adsorption to bare silica as a function of pH shows maximum adsorption at its isoelectric point (pI of pH 8.5) consistent with literature. The desorption rate constants are determined to be ≈10(-5) s(-1), consistent with previously reported values, thus suggesting that shear forces in the capillary may not have a considerable effect on desorption. We anticipate that this study will allow ES-DMA to be used as a "label-free" tool to study adsorption of oligomeric and multicomponent protein systems onto fused silica as well as other surface modifications.     

Determination of sulfadimethoxine in milk with aptamer‐functionalized Fe3O4/graphene oxide as magnetic solid‐phase extraction adsorbent prior to HPLC

An aptamer (Apt) functionalized magnetic material was prepared by covalently link Apt to Fe₃O₄/graphene oxide (Fe₃O₄/GO) composite by 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride and N‐hydroxysuccinimide, and then characterized by FTIR spectroscopy, X‐ray diffraction, and vibration sample magnetometry. The obtained composite of Fe₃O₄/GO/Apt was employed as magnetic solid‐phase extraction adsorbent for the selective preconcentration of sulfadimethoxine prior to analysis by high‐performance liquid chromatography. Under the optimal conditions (sample pH of 4.0, sorbent dosage of 20 mg, extraction time of 3 h, and methanol‐5% acetic acid solution as eluent), a good linear relationship was obtained between the peak area and concentration of sulfadimethoxine in the range of 5.0 to 1500.0 µg/L with correlation coefficient of 0.9997. The limit of detection (S/N = 3) was 3.3 µg/L. The developed method was successfully applied to the analysis of sulfadimethoxine in milk with recoveries in the range of 75.9‐92.3% and relative standard deviations less than 8.1%. The adsorption mechanism of Fe₃O₄/GO/Apt toward sulfadimethoxine was studied through the adsorption kinetics and adsorption isotherms, and the results show that the adsorption process fits well with the pseudo‐second‐order kinetic model and the adsorbate on Fe₃O₄/GO/Apt is multilayer and heterogeneous.