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PubMed1993

A study of the effects of lead foil in dental X-ray film packets on radiographic image quality.

The role of lead foil in protecting film from image degradation due to back-scattered radiation was studied. The effects of low-scattering (polystyrene foam) and tissue-equivalent (Lucite) materials on relative speed and modulation transfer factor (MTF) of the front and back films from Kodak DF-57 double film packets were evaluated. The relative speed of the back film with polystyrene foam behind the film was about 10% faster with lead foil. However, when Lucite was placed behind the film, lead foil eliminated the effect of scattered radiation on film density, MTF, was decreased by the presence of lead foil when polystyrene foam was placed behind the film. With Lucite, however, lead foil improved the MTF value, which was decreased by the scattered radiation at spatial frequencies over 5 cycle mm-1. These results suggest that lead foil itself causes scattered or secondary radiation and affects film speed and resolution. However, it also protects the film from scattered radiation from tissue behind the film and results in improvement in resolution.

Analysis of Variance↗
PubMed2005

Formation of embryoid bodies by mouse embryonic stem cells on plastic surfaces.

Mouse embryonic stem (ES) cells were cultured on artificial polymeric biomembranes with a phospholipid polymer (phosphatidylcholine, PC) surface. ES cells aggregated to form an embryoid body (EB) on the PC surface immediately after seeding. Single EBs formed on the PC surface after 3 d, and their size was depended on the initial number of cells that were seeded. In contrast, many small EBs with a nonuniform shape formed on a conventional hydrophobic nontreated polystyrene surface. RT-PCR assays of the EBs indicated that cell-cell interactions were enhanced in EBs that formed on the PC surface compared with EBs that formed on the polystyrene surface. The transcription factor Pax6, which is a marker of the differentiation of ES cells to neurons, was not expressed in EBs that formed on the PC surface; however, EBs that formed on the polystyrene surface did express Pax6, indicating that they were undergoing differentiation into neurons. When stimulated with retinoic acid (an inducer of differentiation into neurons), EBs on the PC surface expressed Pax6. We also observed that the adhesion of ES cells to the PC surface was reduced. Thus, the formation of large EBs on the PC surface was due to enhanced cell-cell interaction and inhibition of nonspecific differentiation to neurons.

Animals↗
PubMedDate not supplied

Adsorption-induced antigenic changes and their significance in ELISA and immunological disorders.

The functional properties of 125I-labeled antibodies and antigens adsorbed on polystyrene and silicone were compared to their counterparts immobilized by non-adsorptive methods. Less than 20% of polyclonal (pAb) and 1-2% of monoclonal (mAb) capture antibody equivalents remained functional after adsorption as a monolayer. Survivability circa doubled or was totally rescued, when the same antibodies were immobilized via a streptavidin bridge or by using a first stage polyclonal antiglobulin capture antibody, respectively. Similarly, the antigenicity of bovine IgGs for pAb and mAb anti-IgGs was highest when the IgGs were immobilized via a streptavidin bridge or when secondarily adsorbed to an albumin monolayer. IgGs in these configurations were significantly more antigenic than when directly adsorbed on polystyrene or a silicone elastomer. Similar activity was seen after adsorption on polystyrene or silicone. Interestingly, these IgGs were equally antigenic when denatured and subsequently adsorbed in 6M guanidine-HCl versus adsorption in PBS without prior denaturation. Although many of the above finding on antibodies and antigens could be explained by the greater accessibility of antigenic epitopes or antibody binding sites when molecules are immobilized by some type of underlying molecular layer, we also show that certain mAb and pAbs preferentially recognized allotopes on IgG2a when IgG2a was adsorbed. Furthermore, such antigenicity was highest when IgG2a was adsorbed at low, sub-monolayer concentrations. Finally, we show that differences in antigenicity need not be related to the method of immobilization, but can also result from differences in the microenvironment of the epitope. This was demonstrated using a filamentous phage clone specific for fluorescein (FLU). This clone recognizes the fluorescein hapten differently depending on the carrier protein used and the method of conjugation. Data presented in this report indicate that antibodies and antigens adsorbed on hydrophobic polymers undergo changes in their functional properties. Data suggest that both changes in conformation and the accessibility of antigen epitopes or antibody binding sites, most likely occur. Especially in the case of the latter, the functional concentration may be 1-2 orders of magnitude lower than the antibody protein concentration. These observations have implications for immunodiagnostics and emphasize the need to determine the specificity of an antibody in the assay in which it is employed and to make no assumptions about the behavior of solid-phase antigens and antibodies from their behavior in solution. Our studies are also relevant to the use of silicone medical prostheses. The antigenicity of IgGs adsorbed on silicone as a multilayer (secondary layer) is much higher than when directly adsorbed. Since such surfaces would be exposed to very high protein concentrations in vivo, multilayers not a monolayer, would be expected. Thus it would seem from these studies that host protein adsorbed on silicone would be expressed to the immune system at the surface of multilayers. This being the case, it seems unlikely that the adsorption of host protein in vivo would generate new epitopes against which the host's immune system could respond and subsequently initiate an autoimmune syndrome.

Adsorption↗
PubMed1997

Methods used to decrease lithium absorption or enhance elimination.

OBJECTIVE: To review current methods, well documented and investigational, being used to decrease lithium absorption or enhance lithium elimination. METHODS: The basic science and clinical literature on lithium were reviewed by a comprehensive Medline search from 1984 to 1996. Additional references were identified by reviewing the reference citations from the results of the Medline search. RESULTS: Prevention of Absorption: Whole bowel irrigation has been demonstrated to be an effective means of enhancing lithium removal from the gastrointestinal tract. Sodium polystyrene sulfonate resin administration has been shown to be effective in binding lithium elimination in animal and human models. However, the lower limits of effective sodium polystyrene sulfonate dosing and the extent of potassium lowering in humans are questions that need to be answered before sodium polystyrene sulfonate resin can be recommended for routine, use. Enhancement of Elimination: Saline or forced diuresis is not effective in enhancing lithium elimination unless the patient is volume or sodium depleted. The use of continuous arteriovenous hemodiafiltration or low dose dopamine to enhance lithium elimination has only been documented in case reports. Intravenous aminophylline (theophylline) is not consistently effective and its risks outweigh possible benefits. The literature supports hemodialysis as a well documented and effective means of enhancing lithium elimination. Controversy exists over the appropriate indications for its initiation. DISCUSSION: Given the wide interpatient variability in lithium pharmacokinetics, single case reports do not provide sufficient evidence of the effectiveness of a given method to enhance

Animals↗
PubMed1999

Can vector control play a useful supplementary role against bancroftian filariasis?

A single campaign of mass treatment for bancroftian filariasis with diethylcarbamazine (DEC) in Makunduchi, a town in Zanzibar, United Republic of Tanzania, combined with elimination of mosquito breeding in pit latrines with polystyrene beads was followed by a progressive decline over a 5-year period in the microfilarial rate from 49% to 3%. Evidence that vector control had contributed to this long-term decline was obtained by comparison with another town, Moga, where a DEC campaign was used without vector control and where resurgence of microfilariae could be observed 3-6 years after the campaign. In Zanzibar town, treatment of 3844 wet pit latrines and cesspits with polystyrene beads reduced the adult mosquito population in houses by about 65%. Supplementary treatment of open drains and marshes with Bacillus sphaericus produced little or no additional reduction compared to a sector of the town where only pit treatment with polystyrene was carried out. The cost and effort of achieving the 65% reduction in mosquito population could hardly be justified for its impact on filariasis alone, but its noticeable impact on biting nuisance might help to gain community support for an integrated programme.

Animals↗
PubMed1976

[The adhesion of culture cells to some polymers (in vitro)].

Mouse L fibroblasts were grown on such plastic coated coverslips as polyvinyl acetate, cellulose, polymethylmethacrylate, polystyrene and polyvinyl alcohol in in vitro condition, in order to investigate cell contact to various polymeric substrata. Rates of cell multiplication in the presence of the plastics were obtained at the beginning of the experiments, the 2nd day, the 4th day and the 7th day of incubation. Morphological observation in the presence of the plastics were made for 14 hours. Rates of cell multiplication showed no change between most of the experimental groups, i.e., polyvinyl acetate, cellulose, polymethylmethacrylate and polystyrene and control, except polyvinyl alcohol. The cells were deterred from growing with polyvinyl acetate and completely deteriorated on the 4th day of incubation and thereafter. Morphological examination of the adhesion of culture cells to the polymers was made with an arbitrary classification of cell shapes such as round, bipole, spindle or star, depending upon the forms which the cells showed on the substrata under a light microscope, State of adhesion of the cells is considered to be increased in the ascending order of the four classification, polyvinyl acetate was the best in terms of cell adhesion, followed by cellulose, polymethylmethacrylate and polystyrene. The cells did not develop into the advanced state of adhesion in the presence of polyvinyl alcohol. As plastics have become one of the most important biomaterials in recent years, research on biocompatibility of the material, which can be represented by ascertaining of non toxicity, non irritability or non carcinogenecity and adhesion characteristics, is needed. Information on the above would surely be useful in better use of the material and also in developing new materials.

Cell Adhesion↗
PubMed2002

Effect of human serum albumin on cell attachment of human gingival epithelial cells.

OBJECTIVE: To investigate the effect of human serum albumin (HSA) on cell attachment of human gingival epithelial cells (HGE). METHODS: HGE were primary cultured with keratinocyte serum-free medium (KSFM) and dispase. The cultured cells were immunohistochemically stained by monoclonal anti-pan cytokeratin. MTT test was employed to investigate the influence of HSA on the cell attachment on polystyrene surface. The cell growth curve of HGE which were cultured in KSFM with 50 g/L HSA was observed. RESULTS: The results showed significant decrease in cell numbers within 8 hours after HGE were inoculated, in which the polystyrene surface was preincubated with 50 g/L HSA. But it did not prove to be the case from 10 hours to 24 hours after HGE were inoculated. There were no significant difference within 24 hours in cell numbers between cultured in KSFM with 50 g/L HSA and control. The cell numbers in cell growth curve of HGE in KSFM with and without 50 g/L HSA did not show significant difference. CONCLUSIONS: HSA preincubation on polystyrene were produce inhibitory effect of HGE attachment in early stage.

Cell Adhesion↗
PubMed1975

A rapid and sensitive 125I-fibrin solid-phase fibrinolytic assay for plasmin.

125I-fibrinogen, adsorbed to polystyrene tubes at low ionic strength and treated with thrombin, serves as a substrate for a rapid, convenient, and sensitive test tube assay for plasmin and activators and inhibitors of this enzyme. 125I-labeled digestion products released from the 125I-fibrin-polystyrene matrix are readily separated and quantitated and behave, on gel permeation, in the same manner as plasmin-generated degradation products from an unlabeled conventional fibrin clot. The 125I-fibrin, in probable non-cross-linked form, is firmly bound to the polystyrene and is resistant to nonspecific release, with control (no enzyme) values equivalent to 15.2 ng +/- 1.2 (SD) fibrin (1% of the total bound 125I-fibrin). This fact permits consistent detection of lysis of 30-50 ng 125I-fibrin, which exceeds published sensitivities (1000-5000 ng) using 125I- or fluorochrome-labeled fibrin clots as substrate. The sensitivity for plasmin (0.2 mug/ml) is tenfold greater than that of the fibrin-plate method (2.0-2.5 mug/ml), while sensitivities for streptokinase and urokinase activation of plasmin are 0.02 U/ml and 0.04 CTA U/ml, respectively (sensitivity of fibrin-plate method, 0.5 U/ml for both). The method provides a reasonable analogue of the solid-phase nature of fibrin under physiologic conditions, and the ease of preparation of large batches of tubes makes the method suitable for large-scale screening of factors modulating the plasminogen-plasmin system.

Aminocaproates↗
PubMed1992

Comparison of actin changes and calcium metabolism in plastic- and fibronectin-adherent human neutrophils.

Human neutrophils adherent to a polystyrene plastic surface are vigorously activated, whereas those adherent to fibronectin manifest only a priming response. The basis of these metabolic differences was further characterized; polystyrene-adherent cells, which were shown to spread quickly upon adhesion, exhibited an increase of cytoskeleton-associated actin (F-actin) (measured by a nitrobenzoxadiazole-phallacidin fluorescent staining assay) and a decrease of monomeric G-actin concentration (measured by a DNase inhibition assay); in contrast, fibronectin-adherent cells exhibited little spreading and decreased their F-actin, after 1.5 min of adhesion, to 33.49 +/- 6.9% (mean +/- SD, n = 5) of initial levels found in suspended cells before plating. Actin depolymerization in fibronectin-adherent cells was confirmed by measuring G-actin, which sharply increased during the first minute of adhesion, rising from 0.065 +/- 0.007 to 0.20 +/- 0.035 microgram/microgram of protein (mean +/- SEM, p less than 0.05), and then remained elevated during 5 min of observation. In contrast, soluble fibronectin induced a decrease of G-actin in suspended cells. Cells pretreated with 1 microM cytochalasin D and allowed to adhere to a plastic surface did not spread, failed to generate O2-, and exhibited elevated concentrations of G-actin (0.1 to 0.2 microgram/microgram of protein) during the 5 min of observation. Actin changes, as well as respiratory burst, in adherent cells were shown to proceed through a pertussis toxin-insensitive pathway. Fluo-3 measurements of intracellular Ca2+ concentrations ([Ca2+]i) showed a fourfold and twofold [Ca2+]i increase in polystyrene- and fibronectin-adherent cells, respectively, after 2 min. The small rise in [Ca2+]i in fibronectin-adherent cells corresponds to a primed response of these cells to subsequent activation with FMLP. Ionomycin (1 microM) added to neutrophils just before adhesion on fibronectin induced full activation, i.e., O2- production and actin polymerization. The metabolic events controlling metabolic priming and actin depolymerization are as yet uncharacterized, but fibronectin receptor-linked responses beyond the mediation of cell adhesion have now been identified, suggesting complex metabolic functions of integrin receptors.

Actins↗
PubMed1991

Electrophoretic size separation of particles with diameters in the micron range, using polymer solutions.

Polystyrene-sulfate particles ranging in size from 536 to 2,170 nm diameter were subjected to electrophoresis (10 V cm-1, 25 degrees C) in K-MES, 0.03 M ionic strength, pH 6.12, 50 mM CHAPS in liquid polymer media contained in horizontal glass tubes of 1 mm ID. The polymer media were linear polyacrylamide [0.3 to 0.9% Mr = 5 x 10(5) and 5 x 10(6), as well as a commercial solution of the latter in 4 M urea, 7.5% Na2SO4 designated as Gelamide-250] or polyvinylalcohol (0.25 to 2.5%, Mr = 6.5 x 10(5), designated PVA). In these polymer solutions, the polystyrene sulfate particles exhibit linear Ferguson plots [log(mobility) vs polymer concentration]. Their slope, KR, is directly related to the diameter of the particle when electrophoresis is conducted in Gelamide-250 or slowly 25 degrees C solubilized PVA solutions, indicating a molecular sieving mechanism. By contrast, KR is inversely related to the particle diameter when electrophoresis is conducted in linear polyacrylamide of 5 x 10(6) molecular weight or in PVA rapidly solubilized by autoclaving (121 degrees C, 1.2 kg cm-2 pressure), suggesting a particle exclusion (gel permeation) mechanism of size separation. Electrophoresis in solutions of polyacrylamide of 5 x 10(5) molecular weight exhibits the same degree of retardation for the entire size range of polystyrene particles used, i.e. a viscosity effect only and no size separation. Retardation of electrophoretic migration by either a sieving or a permeation mechanism is highly reproducible in polyacrylamide solutions, but not in PVA solutions (whether solubilization conditions favoring an apparent permeation mechanism or a sieving mechanism are applied).(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylic Resins↗
PubMedDate not supplied

Toxigenicity and adherence in Clostridium difficile strains isolated from patients with and without diarrhoea.

The mechanisms by which Clostridium difficile causes diarrhoea are unknown. The expression of putative virulence factors by 44 Clostridium difficile strains isolated from patients with and without diarrhoea was studied. Toxins A and B were tested in CHO and MRC-5 cells, respectively; adherence was measured in two substrates: HEp-2 cells and polystyrene plates. The in vitro expression of toxins A and B by strains isolated from patients with diarrhoea was not significantly different from that by strains isolated from patients without diarrhoea. The ability of adherence to both HEp-2 cells and polystyrene by strains isolated from patients with diarrhoea was not significantly different when compared with strains isolated from patients without diarrhoea; however, strains isolated from adults with diarrhoea seem to adhere to a greater extent to both substrates than strains isolated from adults without diarrhoea. Twenty three strains which did not produce toxins A and B were tested for enterotoxicity in rat small intestine. Eight such strains induced fluid accumulation and seven of them were isolated from children. Adherence to cells and to polystyrene might be an important virulence factor in strains causing diarrhoea in adults; whereas the production of toxins other than A and B might be an important pathogenic mechanism in strains causing diarrhoea in children.

Adult↗
PubMed1991

[Effect of subinhibitory concentrations of beta-lactams on the production of slime, surface hydrophobicity and adhesion of Staphylococcus epidermidis].

The activity of subinhibitory concentrations of oxacillin, imipenem, cefalotin, cefuroxime, cefotaxime and amoxycillin-clavulanic acid upon slime production, surface hydrophobicity and polystyrene adherence of S. epidermidis was evaluated. Slime production and polystyrene adherence of three Staphylococcus epidermidis strains was assessed using test tube (safranin stained film) or in microtiter plate. The surface hydrophobicity was determined using a biphasic system (water/p-xylene). Using one-half and one-quarter of MIC for all antibiotics tested, a reduction in slime production and polystyrene adherence was observed in all three strains used. Regarding surface hydrophobicity, in one of the strains used we have observed a rise in surface hydrophobicity values as compared to control strains whenever one-half of MCI was used for all six antimicrobial agents and also with one-quarter of amoxicillin-clavulanate MCI. Subinhibitory MCI of all six antibiotics tested inhibits slime production and adherence of Staphylococcus epidermidis. This inhibitory effect is not related to changes in surface hydrophobicity.

Anti-Bacterial Agents↗
PubMed1987

Influence of surface chemistry of the substrate on the adsorption of Escherichia coli.

The adsorption of Escherichia coli as influenced by the surface chemistry of different absorbents was studied with microbeads of (1) polystyrene, hydroxymethylated, chloromethylated and aminomethylated polystyrene, (2) cation and anion exchange resins, (3) glass and siliconized glass using a shake test. The adsorption kinetics can be described by a first order rate law for all materials. The specific surface free energy is not even a rough correlation parameter for the adsorption effect. Polar groups with the exception of those with a negative charge (cation exchange resin) favour adsorption of Escherichia coli, but the type of the polar surface group plays a dominating role. Adsorption was maximum with protonated aminomethylated polystyrene which is attributed to the interaction of the positive charge with the negatively charged bacterial surface.

Bacterial Adhesion↗
PubMed1987

Surface modification with protein A for uniform binding of monoclonal antibodies.

We describe optimal conditions for immobilization of two monoclonal antibodies to progesterone for solid-phase assays. Polystyrene surfaces are refined with Protein A to achieve uniform, reproducible, stable, and sterically accessible immobilization of immunoglobulins (IgG). To this end, we optimized the amount of immobilized Protein A, the pH of the medium for immobilization, the concentration of antibody, and the polystyrene surface. We also investigated three carriers for solid-phase assays: 12 X 75 mm polystyrene test tubes, Macrowells (Skatron, Inc.; suitable for processing with multiple pipettors), and microwell strips (Immulon II, Dynatech Inc.). Immunoglobulin does not appreciably dissociate from any of these solid matrices, even if the assay procedure takes several hours. Therefore, we postulate that more than one molecule of immobilized Protein A binds to IgG, or that there is an additional interaction between the antibody and the polymer surface.

Animals↗
PubMed1995

Endothelial cells adherent to expanded polytetrafluoroethylene express the intercellular adhesion molecule-1.

BACKGROUND: The foreign body reaction caused by implanted biomaterials is not fully characterized. To evaluate the effect of an expanded polytetrafluoroethylene (ePTFE) surface on the binding of monoclonal antibodies against the intercellular adhesion molecule (ICAM-1) by adherent endothelial cells, an in vitro bioassay was developed. STUDY DESIGN: Human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC) were grown to confluency on fibronectin-pretreated ePTFE and on fibronectin-pretreated polystyrene culture flasks used as controls. Cells were assayed for size, density, viability, and binding of anti-ICAM-1 antibodies. Human peripheral blood leukocytes (PBLs) were then cocultured with HUVEC adherent to ePTFE and control polystyrene and HUVEC were assayed for the binding of anti-ICAM-1 antibodies. RESULTS: The adherence of HSVEC and HUVEC to ePTFE resulted in a twofold increase in the binding of monoclonal antibodies against ICAM-1 compared with controls at 48 hours (p < 0.005). The addition of PBLs to the adherent HUVEC resulted in a four- to eightfold increase in anti-ICAM-1 antibody binding on ePTFE and polystyrene, respectively, at 24 hours. CONCLUSIONS: Adherence of endothelial cells to ePTFE results in an increase in the binding of anti-ICAM-1 antibodies. Coculture of adherent HUVEC and PBLs results in a marked increase in the number of cells binding ICAM-1 antibodies. These data support the conclusion that ePTFE is associated with the activation of adherent cells, which is one aspect of a multicellular inflammatory response.

Adult↗
PubMed1976

A nonhuman primate vascular shunt model for thrombus generation.

A nonhuman primate model for thrombus generation was developed. Three different types of test devices constructed of polystyrene or polyethylene-Silastic were exposed to flowing blood in arterioarterial or arteriovenous vascular shunts in rhesus monkeys. The test device design included a simple tube, a vortical flow device, and a turbulent flow device. The amount of thrombus deposited within each individual test device after a 15-minute exposure to blood flowing through the shunt was determined gravimetrically. These studies indicate that a test device designed to include an area of vortical flow generated the greatest amount of thrombus. Test devices fabricated from polystyrene consistently generated larger thrombus deposits than did similar test devices fabricated from polyethylene-Silastic. Arteriovenous shunts proved superior to arterioarterial shunts in that flow was predictable in the former and unpredictable in the latter; venovenous shunts thrombosed quickly. Hematologic studies indicated a progressive fall in platelet count during the 4-hour test interval in all animals, whereas in only a few animals were there a shortening of partial thromboplastin time values, a fall in fibrinogen levels, and the appearance of fibrin degradation products. An optimal model for thrombus generation appears to include vortical flow test devices fabricated of polystyrene exposed to flowing blood in an arteriovenous shunt.

Animals↗
PubMed1996

Targeting to intestinal M cells.

The specialised, antigen-transporting, epithelial M cells in the follicle-associated epithelium (FAE) overlying gut-associated lymphoid tissues constitute the primary target for oral delivery of vaccines. Our studies have shown that polystyrene microspheres selectively bind to, and are efficiently transcytosed by, rabbit Peyer's patch M cells in closed intestinal loops. Binding of biodegradable poly(DL-lactide-co-glycolide) microspheres to rabbit Peyer's patch FAE is an order of magnitude lower than that of polystyrene microspheres. Although poly(DL-lactide-co-glycolide) microspheres are not selectively targeted to M cells, a high proportion of those which bind to M cells are transcytosed, supporting the potential of such microspheres as vehicles for oral vaccine delivery. Comparison of the binding of polystyrene microspheres by murine FAE revealed this to be markedly less extensive than by rabbit FAE. These data demonstrate that microsphere binding by M cells depends on the surface properties of both cells and microspheres and suggest that surface modification may enhance the efficacy of microsphere delivery vehicles. One such approach is the incorporation of molecules with inherent binding specificity for M cells. Lectin-binding studies have revealed that M cells exhibit pronounced regional and species variation in glycoconjugate expression. In murine intestine, certain lectins bind selectively to M cells either in Peyer's patches or caecum, or at both sites. Selective targeting to, and transcytosis of, lectin-conjugates by M cells in ligated segments of murine intestine have also been demonstrated. While several lectins display strong selectivity for rabbit caecal M cells, none to date have been identified with specificity for rabbit or rat Peyer's patch M cells. Knowledge of human M cells is limited and no lectin has yet been identified with specificity for these cells. However, at least one lectin exhibits binding specificity for FAE in the human ileum. In the future, knowledge of the regional patterns of M cell carbohydrate expression within a species may allow lectins to be utilised to target selectively antigenic material to the mucosal immune system at specific locations.

Administration, Oral↗
PubMed1999

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.

Avidin↗