Endopolygalacturonase immobilized on epoxide-containing supports

Summary Endopolygalacturonase was immobilized onto 3-(2,3-epoxypropoxypropyl)-silica and oxirane-acrylic beads. Optimum conditions of immobilization and catalytic properties of the immobilized enzyme preparations are described.     

Enzymes immobilized on inorganic supports

Enzymes immobilized on inorganic supports by covalent attachment show unique pH optimums, thermal profiles and kinetics. Organic functional groups are covalently attached to the support using a silane coupling agent. The enzyme may then be covalently coupled to the organic functional groups now on the support. Immobilized enzymes have been characterized and used for several applications.     

Microfluidic electrochemical immunoarray for ultrasensitive detection of two cancer biomarker proteins in serum

A microfluidic electrochemical immunoassay system for multiplexed detection of protein cancer biomarkers was fabricated using a molded polydimethylsiloxane channel and routine machined parts interfaced with a pump and sample injector. Using off-line capture of analytes by heavily-enzyme-labeled 1 μm superparamagnetic particle (MP)-antibody bioconjugates and capture antibodies attached to an 8-electrode measuring chip, simultaneous detection of cancer biomarker proteins prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum was achieved at sub-pg mL?1 levels. MPs were conjugated with ～90,000 antibodies and ～200,000 horseradish peroxidase (HRP) labels to provide efficient off-line capture and high sensitivity. Measuring electrodes feature a layer of 5 nm glutathione-decorated gold nanoparticles to attach antibodies that capture MP-analyte bioconjugates. Detection limits of 0.23 pg mL?1 for PSA and 0.30 pg mL?1 for IL-6 were obtained in diluted serum mixtures. PSA and IL-6 biomarkers were measured in serum of prostate cancer patients in total assay time 1.15 h and sensor array results gave excellent correlation with standard enzyme-linked immunosorbent assays (ELISA). These microfluidic immunosensors employing nanostructured surfaces and off-line analyte capture with heavily labeled paramagnetic particles hold great promise for accurate, sensitive multiplexed detection of diagnostic cancer biomarkers.     

A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins

A fast and robust assay, based on the combination of the highly sensitive immuno-PCR (IPCR), employing standardized self-assembled DNA-protein conjugates as reagents, and the well-established, reliable, and fast real-time PCR detection by means of the TaqMan principle is introduced in this work. The use of anti-species immunoglobulin reagents allows one for easy adaptation of this assay to basically any existing ELISA application. The use of an internal competitor in the real-time IPCR (rtIPCR) further increases the sensitivity and significance of this assay; 0.1-0.01 amol (500-50 fg/mL) IgG from several species (mouse, rabbit, goat, and human) were detectable using direct, indirect, and sandwich model rtIPCR assays, thereby increasing the detection limit of the analogous ELISA tests about 100- to 1000-fold. The robustness of this method was demonstrated in two typical applications by detecting 40 pg/mL of the novel anti-cancer drug rViscumin in human plasma samples as well as 100 pg/mL of a research antibody in cell culture media. In both cases, a comparable ELISA was 1000-fold less sensitive.     

A liposome-PCR assay for the ultrasensitive detection of biological toxins

We describe an ultrasensitive immunoassay for detecting biotoxins that uses liposomes with encapsulated DNA reporters, and ganglioside receptors embedded in the bilayer, as a detection reagent. After immobilization of the target biotoxin by a capture antibody and co-binding of the detection reagent, the liposomes are ruptured to release the reporters, which are quantified by real-time PCR. Assays for cholera and botulinum toxins are several orders of magnitude more sensitive than current detection methods.     

Bioavailable nitrate detection in water by an immobilized luminescent cyanobacterial reporter strain

Cyanobacteria are a major group of photosynthetic bacteria that can accumulate in surface water as so-called "blooms" in response to environmental factors such as temperature, light and certain nutrients such as N, P, and Fe. Some species of cyanobacteria produce toxins, causing a considerable danger for human and livestock health. As a consequence, monitoring of bloom formation and toxin production of drinking water supplies has become a major concern. To enable prediction and monitoring of cyanobacterial blooms, tools to detect nutrient bioavailability in water would be advantageous. A whole-cell biosensor was developed for monitoring nitrate (NO(3-)) bioavailability in aquatic ecosystems using the recombinant bioluminescent cyanobacterial strain Synechocystis PCC 6803 harboring an insertion of a luxAB-kmr fusion with nblA1 in its chromosomal DNA, leading to PnblA::luxAB-kmr. This reporter strain was designated N1LuxKm. Cells were immobilized in microtiter plates and showed a dose-dependent response to nitrate deprivation. The resultant CyanoSensor could detect nitrate in the 4-100 micro M concentration range after a sample incubation time of 10 h under continuous illumination (50 micro E m(-2) s(-1)). The optimal temperature for sensor operation was 29 degrees C and the immobilized biosensor could be stored at 4 degrees C in dark for about 1 month without significant loss of sensitivity.     

Electronmicroscopical detection of iota-carrageenan as its ruthenium red complex

Specific detection of iota-Carrageenan (i-CAR) at the ultrastructural level has been obtained by coupling with ruthenium red (RR) - an electron microscopic stain. The i-CAR-RR complex showed electron density on carbon layers. Peritoneal macrophages were treated with the complex and after 3 h it caused the same morphological changes in macrophages as iota-Carrageenan alone. On the surfaces of macrophages, fine filamentous electron dense material - the i-CAR-RR complex - was detected.     

Electronmicroscopical detection of iota-Carrageenan as its ruthenium red complex

Summary Specific detection of iota-Carrageenan (i-CAR) at the ultrastructural level has been obtained by coupling with ruthenium red (RR) — an electron microscopic stain. The i-CAR-RR complex showed electron density on carbon layers. Peritoneal macrophages were treated with the complex and after 3 h it caused the same morphological changes in macrophages as iota-Carrageenan alone. On the surfaces of macrophages, fine filamentous electron dense material — the i-CAR-RR complex — was detected.     

Distribution and stability of membrane proteins in lipid membranes on solid supports

Bacteriorhodopsin and the nicotinic acetylcholine receptor were biotinylated and reconstituted in lipidic membranes on silicon supports by fusion with proteoliposomes. The presence and distribution of the proteins were studied by binding with streptavidin. Radio-labelled streptavidin was employed for quantifying the amounts of protein remaining in the supported membranes after storage in buffer. The proteins within the membranes remained bound to the surface for weeks. The biological activity of reconstituted unlabelled receptor upon storage showed stability in membranes formed on silicon supports and a reduced stability when formed onto lipid monolayer covered supports. Atomic force microscopy studies on preparations in liquid showed bilayer structures but also attached, partly fused liposomes and membrane particles. In air, the surface was smoother and contained less of liposomes and more of stacked lipid layers. Preparations labelled with streptavidin conjugated to colloidal gold and imaged in air showed the proteins individually distributed, with no protein-rich patches or protein aggregates.     

One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5′-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5′-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5′-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5′-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers.     

A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels

We have simplified the highly sensitive silver stain of R. C. Switzer III, C. R. Merril, and S. Shifrin (1979, Anal. Biochem.98, 231–237) for visualizing proteins in polyacrylamide gels. We have reduced the number of steps in the procedure from 10 to 6, simplified the reagents in each step, and reduced the amount of silver required by a factor of 10, thus greatly reducing the expense of the procedure. In common with the original silver stain, our procedure is 100 times more sensitive than Coomassie brilliant blue and is comparable in sensitivity to radioautography of radioactively labeled proteins.     

Fractionation of membrane proteins on immobilized lectins by high-performance liquid affinity chromatography

Detergent-solubilized glycoproteins were fractionated on high-performance affinity columns employing A concanavalin and Pisum sativum agglutinin as ligands immobilized on microparticulate silica via a propyl spacer. The separations were characterized by high recovery (90-95%) and high specificity (enrichment factor 6- and 58-fold for the bound fractions on A concanavalin and P. sativum agglutinin columns, respectively, compared with the crude extract), as estimated by enzyme-linked lectin assay and chromatographic criteria. In addition, the short running times (30-40 min) make this method highly useful for a first characterization of complex glycoprotein samples and of individual molecules.     

Improved nonporous magnetic supports for immobilized enzymes.

Ni powders coated by deposition of TiO2 or controlled oxidation to NiO develop substantial resistance to corrosion. Chymotrypsin immobilized to these coated Ni supports shows very high stability of activity on storage. Chymotrypsin immobilized by adsorption and glutaraldehyde crosslinking was fairly rapidly eluted under operational conditions in the presence of substrate. If 3-aminopropyltriethoxysilane (APS) was used to produce a covalent linkage, desorption of enzyme still occurred because of relatively unstable bonding of the silane to the oxide surface. A more stable attachment was produced by joining together many silane links with a layer of polyglutaraldehyde. The mechanism of action of APS as a coupling agent under these conditions is discussed. gamma-Fe2O3, and particularly a Mn-Zn ferrite, are suitable magnetic support materials available with smaller particle sizes. Particles below 1 mum give the expected higher specific activities of immobilized enzymes.     

Magnetic supports for immobilized enzymes and bioaffinity adsorbents

Magnetic separation of immobilized enzymes or bioaffinity adsorbents allows their selective recovery from liquors containing other suspended solids, and gives easier handling of large numbers of samples in analysis. Non-porous magnetic supports seem to be more resistant to fouling, diffusional limitation and attrition than conventional porous supports. A variety of magnetic powders and linkage methods have been used in the preparation of supports, though it is not clear how many of these have the required properties for potential applications (including linkage stability, particle size and magnetic properties). Non-porous magnetic supports are attractive for use in liquors containing fouling materials or suspended solids, either for bioaffinity adsorbents or for immobilized enzymes acting on small molecular substrates. Magnetic supports also offer considerable advantages in analysis based on bioaffinity interactions, such as immunoassay.     

Outer membrane complex proteins of Chlamydia pneumoniae

Abstract The protein composition of the outer membrane complex (OMC) of Chlamydia pneumoniae strain AR-39 was analyzed by metabolic labeling with [³⁵S]methionine and [³⁵S]cysteine. Cysteine-rich proteins with molecular masses of 98, 60 doublet, 39.5 (MOMP) and 15.5 kDa were found in the OMC of C. pneumoniae . The cysteine-rich proteins of the OMCs of the threee Chlamydia species showed specific reaction patterns by immunoassay and autoradiography to rabbit or turkey immune sera. Recognition of the MOMP and 60-kDa proteins of the three species was cross-reactive. However, the C. pneumoniae 98-kDa protein was recognized by anti- C. pneumoniae (AR-39) and anti- C. psittaci (TT3) immune sera. None of the immunee sera recognized the 12-kDa cysteine-rich complex.     

Multi-wall carbon nanotube-polyaniline biosensor based on lectin-carbohydrate affinity for ultrasensitive detection of Con A

Surface plasmon resonance (SPR) biosensor detects intracellular signaling events as a change of the angle of resonance (AR). We previously reported that the activation of epidermal growth factor receptor (EGFR) on keratinocytes causes a unique triphasic change of AR, whereas the activation of other receptors, such as IgE receptor and adenosine A3 receptor on mast cells, causes a transient monophasic increase of AR. To study the mechanism of AR changes induced by EGFR activation, we introduced wild and mutated EGFR cDNAs into Chinese hamster ovary (CHO) cells and analyzed changes of AR in response to EGF. CHO cells expressing wild-type EGFR showed a triphasic change of AR, whereas cells expressing kinase-dead EGFR (K721M) showed minimum change of AR. A phosphatidylinositol 3-kinase inhibitor, wortmannin, attenuated the third phase of AR change in CHO cells expressing wild-type EGFR. The pattern of AR change was independent on the concentration of EGF. We also analyzed changes of AR with a nontumorigenic keratinocyte cell line, HaCaT, and several cell lines of carcinoma to explore the feasibility of SPR biosensor as a tool for clinical diagnosis. The activation of HaCaT cells and one out of six carcinoma cell lines showed a full triphasic change of AR. In contrast, five out of the six cell lines showed mono- or bi-phasic change of AR. These results suggest that EGF induces the SPR signals via the phosphorylation of EGFR, and provide a possibility that the SPR biosensor could be applied to the real-time detection and diagnosis of malignant tumors.