Evaluation of antibody immobilization methods for piezoelectric biosensor application

The immobilization of anti-Salmonella antibodies by two methods were studied and evaluated for their potential use in a piezoelectric biosensor. The optimum temperature-time combinations for the highest immobilization yields were determined for both methods. Protein A binding was found to be 67.4+/-3.8% on the gold surface which then allowed an immobilization of 42.1+/-2.09% antibody. The degree of antibody immobilization via surface aldehyde groups of glutaraldehyde (GA) on a precoated quartz crystal with polyethylenimine (PEI) was 31.6+/-0.3%. A piezoelectric probe was designed and used in dry assays to observe the frequency change due to addition of mass by the immobilization layers. The frequency changes recorded showed a better reproducibility and less added mass for the Protein A method. The frequency decrease due to microg of added antibodies was compared to frequency decrease calculated by the Sauerbrey equation. The experimental data was found to be only approximately 8% of theoretical data. The functionality of the immobilized antibodies with the Protein A method was tested with S. typhimurium in a wet chamber and the frequency decrease was compared to results of a similar system activated with PEI-GA immobilization. The frequency decreases with S. typhimurium concentration of approximately 1.5 x 10(9) CFU/ml were 50+/-2 Hz and 44+/-3 Hz for the Protein A method and PEI-GA method, respectively. It was concluded that although both methods resulted in comparable activities in terms of % immobilized protein and frequency decreases due to Salmonella binding, the Protein A method was favorable due to stability and better reproducibility of the immobilization layers.     

Covalent-bonded immobilization of lipase on poly(phenylene sulfide) dendrimers and their hydrolysis ability

Covalent-bonded immobilization of lipase from burkholderia cepacia onto two poly(phenylene sulfide) (PPS) dendrimers with different generations (two and three) was achieved using carbodiimide as a coupling reagent. The hydrolysis activity of olive oil to fatty acid was studied on enzyme-immobilized PPS dendrimers. Enzyme activity was proportional to the enzyme loading, and highest recovered activity was obtained at the medium enzyme loading for both G2 and G3 dendrimers. The immobilization improved the optimum pH and caused the temperature range to widen. Immobilization of enzyme has enhanced the thermal stability of enzyme activity in comparison with free enzyme. The immobilized enzyme as a biocatalyst for batch hydrolysis of olive oil retained 80 approximately 90% activity even after 20 times of recycling. This retention of activity after recycle is very valuable and powerful in enzyme technology. The present noteworthy and vital availability on enzyme reaction of the covalently bonded immobilized lipase on dendrimer came from the structure of dendrimer with a large number of functional terminal groups, which are easily available for immobilization of many lipases at the situation keeping reactive enzymes on the surface of dendrimer.     

桔梗多糖的提取与纯化

中药桔梗饮片经水浸提、乙醇分步沉淀,获得乙醇终浓度60%和80%的两个粗多糖组分,分别命名为PPS60和PPS80。经Molish反应、Fehling试剂反应呈阳性,碘-碘化钾反应呈阴性,说明PPS60和PPS80中均含有多糖和还原糖,但不含有淀粉。运用苯酚-硫酸法测得PPS60和PPS80的总糖含量分别为82.25%和84.17%;3,5-二硝基水杨酸（DNS）法测得还原糖含量分别为6.21%和8.05%。PPS60和PPS80经过Sevage法除蛋白、透析、浓缩和Sephrose 6B凝胶柱层析获得两个单一组分。这些结果为进一步研究桔梗多糖奠定了基础。     

Magnetic immobilization of bacteria using iron oxide nanoparticles

Bacterial cell immobilization is a novel technique used in many areas of biosciences and biotechnology. Iron oxide nanoparticles have attracted much attention in bacterial cell immobilization due to their unique properties such as superparamagnetism, large surface area to volume ratio, biocompatibility and easy separation methodology. Adhesion is the basis behind many immobilization techniques and various types of interactions determine bacterial adhesion. Efficiency of bacterial cell immobilization using iron oxide nanoparticles (IONs) generally depends on the physicochemical properties of the IONs and surface properties of bacterial cells as well as environmental/culture conditions. Bacteria exhibit various metabolic responses upon interaction with IONs, and the potential applications of iron oxide nanoparticles in bacterial cell immobilization will be discussed in this work.     

Immobilization of immunoglobulin-G-binding domain of Protein A on a gold surface modified with biotin ligase

Protein A from Staphylococcus aureus specifically binds to the Fc region of immunoglobulin G (IgG) and is widely used as a scaffold for the immobilization of IgG antibodies on solid supports. It is known that the oriented immobilization of Protein A on solid supports enhances its antibody-binding capability in comparison with immobilization in a random manner. In the current work, we developed a novel method for the oriented immobilization of the IgG-binding domain of Protein A based on the biotinylation reaction from archaeon Sulfolobus tokodaii. Biotinylation from S. tokodaii has a unique property in that the enzyme, biotin protein ligase (BPL), forms a stable complex with its biotinylated substrate protein, biotin carboxyl carrier protein (BCCP). Here, BCCP was fused to the IgG-binding domain of Protein A, and the resulting fusion protein was immobilized on the BPL-modified gold surface of the sensor chip for quartz crystal microbalance through complexation between BCCP and BPL. The layer of the IgG-binding domain prepared in this way successfully captured the antibody, and the captured antibody retained high antigen-binding capability.     

Novel protein a affinity matrix prepared from two-dimensional protein crystals

In this article, we describe a novel type of affinity matrix which was prepared by covalently binding Protein A to crystalline cell surface layers (S-layers) from the gram-positive Clostridium thermohydrosulfuricum L111-69. S-layers were used in the form of cell wall fragments, which were obtained by breaking whole cells by ultrasonification and removing the cell content and the plasma membrane. In these thimble shaped structures, revealing a size of 1 to 2 mum, the peptidoglycan-containing layer was covered on both faces with a hexagonally ordered S-layer lattice composed of identical glycoprotein subunits. After crosslinking the S-layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immobilization of Protein A. Quantitative determination confirmed that up to two Protein A molecules were bound per S-layer subunit leading to a dense monomolecular coverage of the immobilization matrix with the ligand.Affinity microparticles were capable of adsorbing lgG from solutions of purified preparations, from artificial lgG-albumin mixtures, and from serum. The amount of lgG bound to affinity microparticles corresponded to the theoretical saturation capacity. Under appropriate conditions, up to 95% of the adsorbed lgG could be eluted again. Affinity microparticles were found to have an extremely low Protein A leakage and a high stability toward mechanical forces. Because pores in the S-layer lattice revealed a size of 4 to 5 nm, immobilization of Protein A and adsorption of lgG was restricted to the outermost surface area. This excludes mass transfer problems usually encountered with affinity matrices prepared from amorphous polymers where more than 90% of the ligands are immobilized in the interior. (c) 1994 John Wiley & Sons, Inc.     

Detection of estradiol at an electrochemical immunosensor with a Cu UPD|DTBP-Protein G scaffold

A copper monolayer was formed on a gold electrode surface via underpotential deposition (UPD) method to construct a Cu UPD|DTBP-Protein G immunosensor for the sensitive detection of 17β-estradiol. Copper UPD monolayer can minimize the non-specific adsorption of biological molecules on the immunosensor surface and enhance the binding efficiency between immunosensor surface and thiolated Protein G. The crosslinker DTBP (Dimethyl 3,3'-dithiobispropionimidate · 2HCl) has strong ability to immobilize Protein G molecules on the electrode surface and the immobilized Protein G provides an orientation-controlled binding of antibodies. A monolayer of propanethiol was firstly self-assembled on the gold electrode surface, and a copper monolayer was deposited via UPD on the propanethiol modified electrode. Propanethiol monolayer helps to stabilize the copper monolayer by pushing the formation and stripping potentials of the copper UPD monolayer outside the potential range in which copper monolayer can be damaged easily by oxygen in air. A droplet DTBP-Protein G was then applied on the modified electrode surface followed by the immobilization of estradiol antibody. Finally, a competitive immunoassay was conducted between estradiol-BSA (bovine serum albumin) conjugate and free estradiol for the limited binding sites of estradiol antibody. Square wave voltammetry (SWV) was employed to monitor the electrochemical reduction current of ferrocenemethanol and the SWV current decreased with the increase of estradiol-BSA conjugate concentration at the immunosensor surface. Calibration of immunosensors in waste water samples spiked with 17β-estradiol yielded a linear response up to ≈ 2200 pg mL(-1), a sensitivity of 3.20 μA/pg mL(-1) and a detection limit of 12 pg mL(-1). The favorable characteristics of the immunosensors such as high selectivity, sensitivity and low detection limit can be attributed to the Cu UPD|DTBP-Protein G scaffold.     

Effect of pentosan polysulphate, standard heparin and related compounds on protein kinase C activity.

Ca2+/phospholipid-dependent protein kinase (PKC) was inhibited by sulphated polysaccharides. Pentosan polysulphate (PPS) and heparin were 8-10-times more potent than dextran sulphate or heparan sulphate. Steady-state studies revealed that PPS was a competitive inhibitor with respect to ATP with an apparent Ki value of 0.32 micrograms/ml and a non-competitive inhibitor with respect to histones. In contrast, the inhibition of PKC by heparin was competitive with substrate and non-competitive with respect to ATP. The interaction of sulphated polysaccharides with the catalytic domain of PKC was further demonstrated by the absence of effect on [3H]phorbol 12,13-dibutyrate binding to the regulatory domain of PKC. Furthermore, PPS and heparin inhibited equally cAMP-dependent protein kinase and tyrosine protein kinase. Structure-function relationships indicated that the Inhibition of protein kinases by PPS and heparin fractions was highly dependent on molecular weight. Additionally, PKC-affinity chromatography revealed that a high-molecular-weight heparin fraction with strong anti-PKC activity was eluted. We set out to demonstrate that heparin and PPS, which are potent antiproliferative agents on vascular smooth muscle cells (SMC), alter intracellular PKC activity (both membrane and cytosolic). Therefore, it is suggested that the mechanism by which sulphated polysaccharides inhibit SMC growth may be by direct inhibition of PKC in SMC.     

Recent advances in nano-carrier immobilized enzymes and their applications

Enzymes have been widely used because of their catalytic properties, and immobilization is a promising technique to improve their catalytic activity and stability. Due to their large specific surface areas, exceptional chemical, mechanical, thermal and cost effective characteristics, nanomaterials should be ideal carriers for the immobilization of enzymes. Enzymes immobilized on nano-carriers are more robust and stable, and can be recycled and reused. This review focuses on the nanomaterial immobilized enzymes and their applications. The introduction addresses the advantages of immobilized enzymes and the features of enzyme immobilization nanocarriers. The next section covers carbonaceous nanomaterials used in enzymes immobilization, with subsections on carbon nanotube, graphene, graphene oxide and reduced graphene oxide. The third section treats metallic nanomaterials for enzymes immobilization, with subsections on metal (gold), metal oxide (titanium dioxide, zinc oxide) and metal hydroxide (layered double hydroxide) nanomaterials. Then, the next section summarizes the applications of nanomaterial immobilized enzymes. A concluding section discusses the challenges and prospects of nanomaterial immobilized enzymes.     

Factors influencing the production of water-soluble endopolysaccharides and exopolysaccharides from Lentinus lepideus and their effects on immune cytokine production

An efficient method to produce water-soluble polysaccharides from Lentinus lepideus is described. The productivity of both endopolysaccharides (PPS) and exopolysaccharides (EPS) was compared under various culture conditions. The effect of treating their own PPS and EPS on immune cytokine production was also studied in relation to culture factors. High yield production of EPS required moderate culture temperature (25 degrees ) as well as long culture period (16-20 days). In contrast, PPS production required high culture temperature (30 degrees ) and short culture period ( days). Most of the carbon sources did not affect polysaccharides and mycelial production except for sucrose. Immune cytokine levels in the EPS treatment varied among carbon sources or culture periods. PPS did not appear to affect much on the production of cytokines, regardless of the culturing factors, except for the culture period. These results suggest that the optimal culture conditions for L. lepideus vary according to culture purposes, and different culture conditions should be used for different targets including mycelial biomass, EPS, and PPS. Whereas the immunomodulating activity of EPS appeared to be affected by culture conditions in L. lepideus, that of PPS did not.     

Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function

The advantages of oriented immobilization of biologically active proteins are good steric accessibilities of active binding sites and increased stability. This not only may help to increase the production of preparative procedures but is likely to promote current knowledge about how the living cells or tissues operate. Protein inactivation starts with the unfolding of the protein molecule by the contact of water with hydrophobic clusters located on the surface of protein molecules, which results in ice-like water structure. Reduction of the nonpolar surface area by the formation of a suitable biospecifc complex or by use of carbohydrate moieties thus may stabilize proteins. This review discusses oriented immobilization of antibodies by use of immobilized protein A or G. The section about oriented immobilization of proteins by use of their suitable antibodies covers immobilization of enzymes utilizing their adsorption on suitable immunosorbents prepared using monoclonal or polyclonal antibodies, preparation of bioaffinity adsorbent for the isolation of concanavalin A and immobilization of antibodies by use of antimouse immunoglobulin G, Fc-specific (i.e. specific towards the constant region of the molecule). In the further section immobilization of antibodies and enzymes through their carbohydrate moieties is described. Oriented immobilization of proteins can be also based on the use of boronate affinity gel or immobilized metal ion affinity chromatography technique. Biotin–avidin or streptavidin techniques are mostly used methods for oriented immobilization. Site-specific attachment of proteins to the surface of solid supports can be also achieved by enzyme, e.g., subtilisin, after introduction a single cysteine residue by site-directed mutagenesis.     

Impedance-based detection of DNA sequences using a silicon transducer with PNA as the probe layer

Electrochemical impedance measurements were used for the detection of single-strand DNA sequences using a peptide nucleic acid (PNA) probe layer immobilized onto Si/SiO2 chips. An epoxysilane layer is first immobilized onto the Si/SiO2 surface. The immobilization procedure consists of an epoxide/amine coupling reaction between the amino group of the PNA linker and the epoxide group of the silane. A 20-nucleotide sequence of PNA was used. Impedance measurements allow for the detection of the changes in charge distribution at the oxide/solution interface following modifications to the oxide surface. Due to these modifications, there are significant shifts in the semiconductor's flat-band potential after immobilization and hybridization. The results obtained using this direct and rapid approach are supported by fluorescence measurements according to classical methods for the detection of nucleic acid sequences.     

Monitoring the assembly of antibody-binding membrane protein arrays using polarised neutron reflection

Protein arrays are used in a wide range of applications. The array described here binds IgG antibodies, produced in rabbit, to gold surfaces via a scaffold protein. The scaffold protein is a fusion of the monomeric E. coli porin outer membrane protein A (OmpA) and the Z domain of Staphylococcus aureus protein A. The OmpA binds to gold surfaces via a cysteine residue in a periplasmic turn and the Z domain binds immunoglobulins via their constant region. Polarised Neutron Reflection is used to probe the structure perpendicular to the gold surface at each stage of the assembly of the arrays. Polarised neutrons are used as this provides a means of achieving extra contrast in samples having a magnetic metal layer under the gold surface. This contrast is attained without resorting to hydrogen/deuterium exchange in the biological layer. Polarised Neutron Reflection allows for the modelling of many and complex layers with good fits. The total thickness of the biological layer immobilised on the gold surface is found to be 187 A and the layer can thus far be separated into its lipid, protein and solvent parts.     

Improved immunomatrix methods to detect protein:protein interactions

Immunoprecipitation (IP) and coimmunoprecipitation (co-IP) are key techniques for studying protein-protein interactions. These methods utilize immobilized Protein A or Protein G to isolate antibody-bound target antigens. The main disadvantage of traditional IP and co-IP is that the conditions used to elute the precipitated antigen also release the antibody thus contaminating the antigen and destroying the antibody support. To overcome these problems, we describe two methods to generate a reusable antibody support by cross-linking the antibody to immobilized Protein A or Protein G, or by coupling it directly to the resin (see Scheme 1). Antibody cross-linking can be done in 1 h while antibody coupling requires 4 h. IP or co-IP is accomplished by incubating the antibody resin with the protein sample. Washes and elutions are carried out in a spin column to reduce resin loss and decrease assay time. Target proteins are eluted with 0.1 M glycine (pH 2.8) and the resin-bound antibody is re-equilibrated in phosphate-buffered saline (PBS) for reuse. Our studies have demonstrated that the immobilization efficiency for the antibody coupling method was similar for several species of antibody. Furthermore, we illustrate that using both methods of antibody immobilization yield IP and co-IP results similar to traditional protocols but eliminate the antibody heavy and light chain contamination.     

Protein immobilization to alumina supports: I. characterization of alumina-organophosphate ligand interactions and use in the attachment of papain

The chemical adsorption of organic phosphate compounds to alumina has been used to create surface linkers for protein immobilization. A number of particulate alumina supports were screened for their physical properties and ability to bind organic phosphate compounds. Two aluminas, termed C1 and CPC, were selected based on their suitability for subsequent testing as protein immobilization supports. Papain was successfully immobilized to these supports when derivatized with phosphate compounds containing free terminal carboxyl groups. Protein binding was enhanced when support carboxyl groups were activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The level of papain immobilization was dependent upon the length of the linker used and the mass of protein exposed to the support. (c) 1992 John Wiley & Sons, Inc.     

Survival of mycobacteria depends on proteasome‐mediated amino acid recycling under nutrient limitation

Intracellular protein degradation is an essential process in all life domains. While in all eukaryotes regulated protein degradation involves ubiquitin tagging and the 26S‐proteasome, bacterial prokaryotic ubiquitin‐like protein (Pup) tagging and proteasomes are conserved only in species belonging to the phyla Actinobacteria and Nitrospira. In Mycobacterium tuberculosis, the Pup‐proteasome system (PPS) is important for virulence, yet its physiological role in non‐pathogenic species has remained an enigma. We now report, using Mycobacterium smegmatis as a model organism, that the PPS is essential for survival under starvation. Upon nitrogen limitation, PPS activity is induced, leading to accelerated tagging and degradation of many cytoplasmic proteins. We suggest a model in which the PPS functions to recycle amino acids under nitrogen starvation, thereby enabling the cell to maintain basal metabolic activities. We also find that the PPS auto‐regulates its own activity via pupylation and degradation of its components in a manner that promotes the oscillatory expression of PPS components. As such, the destructive activity of the PPS is carefully balanced to maintain cellular functions during starvation.     

Surface functionalization using catalyst-free azide-alkyne cycloaddition

The utility of catalyst-free azide-alkyne [3 + 2] cycloaddition for the immobilization of a variety of molecules onto a solid surface and microbeads was demonstrated. In this process, the surfaces are derivatized with aza-dibenzocyclooctyne (ADIBO) for the immobilization of azide-tagged substrates via a copper-free click reaction. Alternatively, ADIBO-conjugated molecules are anchored to the azide-derivatized surface. Both immobilization techniques work well in aqueous solutions and show excellent kinetics under ambient conditions. We report an efficient synthesis of aza-dibenzocyclooctyne (ADIBO), thus far the most reactive cyclooctyne in cycloaddition to azides. We also describe convenient methods for the conjugation of ADIBO with a variety of molecules directly or via a PEG linker.     

Expressed protein ligation to obtain selectively modified aldo/keto reductases

Specific enzyme immobilization has moved into the focus for many applications in biochemical research fields. Expressed Protein Ligation (EPL) has been proven to be ideal to selectively label proteins at single positions. Applying this technique to enzymes of the aldo/keto reductase superfamily provides a new approach to generate native or modified redox enzymes for direct and indirect immobilization.     

Phenotypic analysis of pneumococcal polysaccharide-specific B cells

The phenotype of B cells responsible for the production of anti-pneumococcal polysaccharide Ab has been unclear. Although individuals that respond poorly to the 23-valent pneumococcal polysaccharide (PPS) vaccine, Pneumovax, such as children <2 y, the asplenic, and a subset of common variable immunodeficiency patients, are profoundly deficient or lack IgM memory cells (CD27(+)IgM(+)), they are also deficient in the switched memory (CD27(+)IgM(-)) compartment. Direct characterization of PPS-specific B cells has not been performed. In this study, we labeled PPS14 and PPS23F with fluorescent markers. Fluorescently labeled PPS were used in FACSAria flow cytometry to characterize the phenotype of PPS-specific B cells obtained from 18 young adults pre- and postimmunization with Pneumovax. The labeled PPS were capable of inhibiting binding of Ab to the native PPS. Similarly, the native PPS were able to inhibit binding of PPS-specific B cells in a flow cytometric assay demonstrating specificity and functionality. Phenotypic analysis of unselected B cells, pre- and postimmunization, demonstrated a predominance of naive CD27(-)IgM(+) cells accounting for 61.5% of B cells. Likewise, the PPS-specific B cells obtained preimmunization consisted primarily of naive, CD27(-) B cells, 55.4-63.8%. In contrast, the PPS-specific B cells obtained postimmunization were predominantly IgM memory cells displaying the CD27(+)IgM(+), 54.2% for PPS14 and 66% for PPS23F, significantly higher than both unselected B cells and PPS-specific B cells. There was no significant difference in switched memory B cell populations (CD27(+)IgM(-)) between groups. These results suggest a dominant role of IgM memory cells in the immune response to pneumococcal polysaccharides.     

A universal protocol for photochemical covalent immobilization of intact carbohydrates for the preparation of carbohydrate microarrays

A universal photochemical method has been established for the immobilization of intact carbohydrates and their analogues, and for the fabrication of carbohydrate microarrays. The method features the use of perfluorophenyl azide (PFPA)-modified substrates and the photochemical reaction of surface azido groups with printed carbohydrates. Various aldoses, ketoses, nonreducing sugars such as alditols, and their derivatives can be directly arrayed on the PFPA-modified chips. The lectin-recognition ability of arrayed mannose, glucose, and their oligo- and polysaccharides were confirmed using surface plasmon resonance imaging and laser-induced fluorescence imaging.