On-chip ultra-thin layer chromatography and surface enhanced Raman spectroscopy

We demonstrate that silver nanorod (AgNR) array substrates can be used for on-chip separation and detection of chemical mixtures by combining ultra-thin layer chromatography (UTLC) and surface enhanced Raman spectroscopy (SERS). The UTLC-SERS plate consists of an AgNR array fabricated by oblique angle deposition. The capability of the AgNR substrates to separate the different compounds in a mixture was explored using a mixture of four dyes and a mixture of melamine and Rhodamine 6G at varied concentrations with different mobile phase solvents. After UTLC separation, spatially-resolved SERS spectra were collected along the mobile phase development direction and the intensities of specific SERS peaks from each component were used to generate chromatograms. The AgNR substrates demonstrate the potential for separating the test dyes with plate heights as low as 9.6 μm. The limits of detection are between 10(-5)-10(-6) M. Furthermore, we show that the coupling of UTLC with SERS improves the SERS detection specificity, as small amounts of target analytes can be separated from the interfering background components.     

Immobilization of antibody fragment for immunosensor application based on surface plasmon resonance

Biosurface fabrication using the Fab′ fragment of immunoglobulin (IgG) was carried out by self-assembly (SA) technique. The pepsin-digested monoclonal antibody (Mab) against bovine insulin containing the F(ab′)₂ fragment and residual proteins was separated using affinity chromatography and dialysis. To prevent the nonspecific binding of F(ab′)₂ onto gold (Au) substrate, the native disulfide bridge was reduced using dithiothreitol (DTT) to convert F(ab′)₂ into Fab′, which made the immobilization to be carried out via the native thiol (–SH) group. The fabricated biosurface using SA technique showed the formation of stable thin film through AFM topography. Through the concentration change of DTT and Fab′, the absorption characteristics against the Au surface were investigated using surface plasmon resonance (SPR) with the flow cell. The amount of immobilized antibody fragment and the antigen binding capacity were regulated with respect to the reduction state and concentration of F(ab′)₂. Based on the biosurface of the fabricated Fab′, the insulin-detection was carried out by the measurement of SPR. The proposed antibody surface could successfully detect the bovine insulin at the concentration from 100 ng/mL to 10 μg/mL.     

Single ether group in a glycol-based ultra-thin layer prevents surface fouling from undiluted serum

Through systematic structural modification, it is shown that the internal, single oxygen atom of simple monoethylene glycol-based organic films is essential for radically altering the fouling behaviour of quartz against undiluted serum, as characterized by the electromagnetic piezoelectric acoustic sensor. The synergy is strongest with distal hydroxyls.     

Probing adsorbed fibronectin layer structure by kinetic analysis of monoclonal antibody binding

Adsorbed protein layers are often away from equilibrium and thus exhibit history dependent structures. We use the kinetics of monoclonal antibody binding, as measured using optical waveguide lightmode spectroscopy (OWLS), to investigate the structure of adsorbed fibronectin (Fn) layers formed under different kinetic paths. For all of the layers investigated, we find no difference between the apparent adsorption rate constants of (i) monoclonal antibodies specific to Fn's cell binding site (alpha-Fn) and (ii) monoclonal antibodies specific to cytochrome c (alpha-CC, as a control), indicating initial adsorption of antibodies to be non-specific. For certain layers, the saturation density and the initial projected area per antibody differ significantly between alpha-Fn and alpha-CC, suggesting specific binding to follow the initial non-specific attachment. The fraction of antibodies binding specifically to the Fn layer, and the number of Fn binding sites per specific binding event, are estimated in terms of the difference in initial projected areas between alpha-Fn and alpha-CC. For a Fn layer formed at a bulk concentration of 2 microg/mL, we find a decrease in specific binding with an increase in Fn layer formation time, suggesting post-adsorption structural changes of a lower density adsorbed layer diminish binding site availability. Conversely, for a Fn layer formed at a bulk concentration of 40 microg/mL, we find an increase in specific binding with an increase in the aging time of the Fn layer, implying post-adsorption structural changes reveal binding sites for a higher density adsorbed layer.     

Electrospun glassy carbon ultra-thin layer chromatography devices

The development and application of electrospun glassy carbon nanofibers for ultra-thin layer chromatography (UTLC) are described. The carbon nanofiber stationary phase is created through the electrospinning and pyrolysis of SU-8 2100 photoresist. This results in glassy carbon nanofibers with diameters of ∼200–350 nm that form a mat structure with a thickness of ∼15 μm. The chromatographic properties of UTLC devices produced from pyrolyzed SU-8 heated to temperatures of 600, 800, and 1000 °C are described. Raman spectroscopy and scanning electron microscopy (SEM) are used to characterize the physical and molecular structure of the nanofibers at each temperature. A set of six laser dyes was examined to demonstrate the applicability of the devices. Analyses of the retention properties of the individual dyes as well as the separation of mixtures of three dyes were performed. A mixture of three FITC-labeled essential amino acids: lysine, threonine and phenylalanine, was examined and fully resolved on the carbon UTLC devices as well. The electrospun glassy carbon UTLC plates show tunable retention, have plate number, N, values above 10,000, and show physical and chemical robustness for a range of mobile phases.     

Aligned electrospun nanofibers for ultra-thin layer chromatography

The fabrication and implementation of aligned electrospun polyacrylonitrile (PAN) nanofibers as a stationary phase for ultra-thin layer chromatography (UTLC) is described. The aligned electrospun UTLC plates (AE-UTLC) were characterized to give an optimized electrospun mat consisting of high nanofiber alignment and a mat thickness of ∼25 μm. The AE-UTLC devices were used to separate a mixture of β-blockers and steroidal compounds to illustrate the properties of AE-UTLC. The AE-UTLC plates provided shorter analysis time (∼2–2.5 times faster) with improved reproducibility (as high as 2 times) as well as an improvement in efficiency (up to100 times greater) relative to non-aligned electrospun-UTLC (E-UTLC) devices.     

Immobilization of several multienzyme systems on porous glass beads

Equimolar concentrations of malate dehydrogenase (EC 1.1.1.37) and fumarase (EC 4.2.1.2) and equimolar concentrations of malate dehydrogenase and citrate synthase (EC 4.1.3.7) were simultaneously immobilized to alkylamine porous silica beads with gluteraldehyde. The activity of each enzyme in the two-enzyme immobilized systems was determined and exact concentrations of the free nonimmobilized enzymes were prepared. The activities of the coupled free and coupled immobilized systems were measured, and it was observed that there was a 10-fold enhancement in the catalysis of the immobilized enzymes.     

Immobilization of antibodies on glass surfaces through sugar residues

This work characterizes substrates for immunoassays obtained through the immobilization of vectorially oriented antibodies on glass. The method of preparation is based on the condensation reaction between an aldehyde group on the F(c) portion of antibodies and the hydrazide group on the modified glass surface. Light microscopy and AFM imaging in height and phase modes were used to assess the properties of the modified surface. Both techniques are consistent with a fairly uniform antibody coverage on the micrometer and submicrometer scales. ELISA tests were used to evaluate the activity and surface distribution of immobilized antibodies as well as nonspecific binding to surfaces after various modification steps. It was shown that exposure of the surfaces to a BSA solution minimized nonspecific binding to undetectable levels.     

Monitoring carbamazepine in surface and wastewaters by an immunoassay based on a monoclonal antibody

The pharmaceutical compound carbamazepine (CBZ) is an emerging pollutant in the aquatic environment and may potentially be used as a wastewater marker. In this work, an enzyme-linked immunosorbent assay (ELISA) for the detection of carbamazepine in surface and sewage waters has been developed. The heterogeneous immunoassay is based on a commercially available monoclonal antibody and a novel enzyme conjugate (tracer) that links the hapten via a hydrophilic peptide (triglycine) spacer to horseradish peroxidase. The assay achieves a limit of detection of 24 ng/L and a quantitation range of 0.05–50 μg/L. The analytical performance and figure of merits were compared to liquid chromatography–tandem mass spectrometry after solid-phase extraction. For nine Berlin surface water samples and one wastewater sample, a close correlation of results was observed. A constant overestimation relative to the CBZ concentration of approximately 30% by ELISA is probably caused by the presence of 10,11-epoxy-CBZ and 2-hydroxy-CBZ in the samples. The ELISA displayed cross-reactivities for these compounds of 83% and 14%, respectively. In a first screening of 27 surface water samples, CBZ was detected in every sample with concentrations between 0.05 and 3.2 μg/L. Since no sample cleanup is required, the assay allowed for the determination of carbamazepine with high sensitivity at low costs and with much higher throughput than with conventional methods.      

Immobilization of polysaccharides on a fluorinated silicon surface

A self-assembled monolayer (SAM) of fluoroalkyl silane (FAS) was deposited on a silicon surface by chemical vapor deposition (CVD) at room temperature under 1.01x10(5)Pa nitrogen. Using this new approach, the quality and reproducibility of the SAM are better than those prepared either in solution or by vapor phase deposition, and the deposition process is simpler. In this modified CVD process, the silane monomers, instead of the oligomeric species, are the primary reactants. Full coverage of the silicon surface by FAS molecules was achieved within 5 min. Heparin and hyaluronan, two naturally occurring biocompatible polysaccharides, were successfully covalently attached on the FAS SAM/Si surface by photo-immobilization. Atomic force microscopy (AFM) revealed the morphologic changes after the immobilization of heparin and hyaluronan, and X-ray photoelectron spectroscopy (XPS) confirmed the change in chemical compositions. Such combination of coatings is expected to enhance the stability and biocompatibility of the base material.     

The stability of ultra-thin perfluoropolyether mixture films on the amorphous nitrogenated carbon surface

The thermodynamic stability of boundary lubricant films based upon mixtures of liquid perfluoropolyethers (PFPEs) is reported. Mixtures of A20H-2000 with Zdols 2000, 2500, and 4000 and Zdol-TX 2200 on amorphous carbon nitride films are investigated. An optical surface analyzer is used to image the autophobic dewetting of the mixture PFPE films. The critical dewetting thickness coincides with the monolayer thickness of the adsorbed mixture PFPE films as determined by the changes in the surface energy as a function of lubricant film thickness. The critical dewetting thickness varies linearly with mixture concentration.     

Immobilization of enzymes on a microchannel surface through cross-linking polymerization

A novel and facile method for the preparation of an enzyme-immobilized microreactor has been developed in which enzymes are immobilized as an enzyme-polymer membrane formed on the inner wall of the microchannel by a cross-linking polymerization method; the resulting microreactor shows excellent reaction performance and stability against denaturating agents.     

Effect of surface packing density of interfacially adsorbed monoclonal antibody on the binding of hormonal antigen human chorionic gonadotrophin

Interfacial adsorption of a mouse monoclonal antibody (type IgG1, anti-beta-hCG) at the hydrophilic silicon oxide/water interface has been studied by spectroscopic ellipsometry and neutron reflection, followed by assessment of binding of a hormonal antigen, human chorionic gonadotrophin (hCG), onto the adsorbed antibody molecules. The amount of adsorption reached a maximum around the isoelectric pH (IP) of 6 for the antibody; this pH-dependent pattern could be altered by increasing salt concentration, a trend also observed for other proteins. Neutron reflection revealed the formation of a 40 A uniform layer from the adsorbed antibody, indicating a flat-on orientation. The subsequent hCG binding showed that the molar ratio of hCG bound to antibody at the interface was as high as 0.7 at low surface coverage of antibody and decreased with increasing surface antibody concentration. The results point to an increasing extent of steric hindrance to hCG access with increasing packing density of antibody molecules on the surface. Comparison with previously published crystal structure studies suggests twisting of the variable region to allow access of the antigen. The binding of hCG was also found to be pH-dependent with its maximum around the IP, if the ionic strength of the solution was low (20 mM). However, if the ionic strength was increased to 200 mM, then hCG binding was influenced by a combination of steric hindrance and electrostatic interaction between the antigen and the surface. These results are highly relevant to the improvement of the performance of biotechnologies such as fertility test pads and biosensors based on antibody immobilization.     

Adsorption of monoclonal antibody variants on analytical cation-exchange resin

Monoclonal antibody (MAb) variants differing by one or two C-terminal lysine residues can be separated by cation-exchange chromatography due to the difference in their charge distribution. The adsorption of the three MAb variants on a weak cation-exchange resin was characterized using directly the raw mixture in spite of the presence of some impurities. The effects of both, pH and eluent salt concentration, on the adsorption isotherm were investigated. Under certain experimental conditions distorted peak shapes and even peak doubling for single variant injections were obtained, in addition to unexpectedly long retention times. These observations were explained based on equilibrium theory. The separation of the MAb variants was designed for an isocratic and a linear salt gradient operation. The corresponding optimal values of pH and salt concentration were determined. The use of salt gradients not only allows reducing the process time and increasing enrichment of the variants, but also leads to some loss in purity. A baseline separation could be obtained under isocratic and strongly adsorbing conditions at pH 6.3. A lumped kinetic model and a procedure for estimating the corresponding parameters were developed and validated by comparison with experimental elution chromatograms in a wide range of operating conditions.     

Enhancement of X-ray fluorescence intensity from an ultra-thin sandwiched layer at grazing-emission angles

Ni ultra-thin films sandwiched with carbon thin films of different thickness are measured by a laboratory grazing-emission X-ray fluorescence instrument. The Ni Kα intensity of the Ni ultra-thin film sandwiched with carbon layers is three times enhanced in comparison with the Ni ultra-thin film without carbon layers. In addition, oscillations caused by interference effects of directly observed X-ray beams and the reflected X-ray beams on the surface of the Pt substrate, are clearly observed. The periods of the oscillations depends on the thickness of the carbon layer, that is, the position of the Ni layer. Therefore, the thickness of the carbon layer can be estimated.     

毛细管电泳技术在单克隆抗体药物分析中的应用

单克隆抗体药物在生物制药行业占有重要地位,是生物医药领域发展的主要方向。因此,单克隆抗体药物的质量控制已成为全球生物制药企业及法规机构关注的热点,对单克隆抗体药物精确表征的需求日益增加。毛细管电泳技术具有分离效率高、分析速度快、分离模式多、样品用量少等特点,已成为单克隆抗体药物分析和质量控制的重要手段。该文对毛细管凝胶电泳、毛细管等电聚焦、毛细管区带电泳等模式在单克隆抗体药物的纯度分析、等电点测定、电荷异质性分析和N-寡糖分析的应用进行综述,以期为国内单克隆抗体研究开发和生产的企事业单位提供技术参考。     

Glutamine deamidation of a recombinant monoclonal antibody

Deamidation of glutamine (Gln) proceeds at a much slower rate than deamidation of asparagine (Asn) residues at peptide level. However, deamidation of Gln residues in native proteins may occur faster because of the impact of protein structure and thus plays a significant role in affecting protein stability. Gln deamidation of a recombinant monoclonal IgG1 antibody was investigated in the current study. Deamidation was determined by a molecular weight increase of 1 Da, a retention time shift on reversed‐phase chromatography and tandem mass spectrometric (MS/MS) analysis of the peptides. As expected, Gln residues at different locations in the three‐dimensional structure had different susceptibilities to deamidation. Gln deamidation was highly pH dependent with the highest level detected in the sample incubated at pH 9, and lowest level at pH 6 in the pH range from 5 to 9. The detection of significant levels of Gln deamidation suggested that it may play an important role in affecting heterogeneity and stability of recombinant monoclonal antibodies. Copyright © 2008 John Wiley & Sons, Ltd.     

Immobilization of urease on the silica gel surface by sol-gel method

The possibility of “double immobilization” of urease on silica gel, involving enzyme incorporation into the polysiloxane polymer prepared by sol-gel procedure on the surface of an inorganic support was studied. The optimal composition and thickness of the polysiloxane film, providing relatively high activity of urease, were determined.