Alumina-phosphate complexes for immobilization of biomolecules

Organic compounds containing the -PO3H2 function are strongly and specifically adsorbed by aluminum oxide in water within a large range of pH. The reversible character of the interaction allows the adsorbed organic phosphates to be displaced by inorganic phosphate buffers resulting in their purification by an affinity-like chromatographic procedure. The interaction between alumina and selected multifunctional compounds containing a phosphonate group yields a chemically activated alumina-phosphate complex onto which enzymes or other molecules can be immobilized. A number of proteases immobilized on alumina through such phosphate interactions proved to be active in the presence of organic solvents. As a consequence, enzyme-catalyzed peptide synthesis in a water-limited environment and optical resolution of amino acids in water-organic solvent emulsions can be accomplished.     

A novel matrix for the immobilization of acetylcholinesterase

In this study, a new matrix for immobilization of acetylcholinesterase was investigated by using alginate and kappa-carrageenan. The effects of pH, temperature, storage and thermal stability on the free and immobilized acetylcholinesterase activity were examined. Maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) was also investigated for free and immobilized enzymes. For free and immobilized enzymes into Ca-alginate and alginate/kappa-carrageenan polymer blends, optimum pH and temperature was found to be 7 and 30 degrees C, respectively. For free enzyme, maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) values were found to be 6.35 mM and 50 mM min(-1), respectively, the same values for immobilized enzymes were determined as 8.68, 12.7 mM and 39.7, 52.9 mM min(-1), respectively. Storage and thermal stability of acetylcholinesterase was increased by as a result of immobilization.     

A novel dry method for surface modification of SU-8 for immobilization of biomolecules in Bio-MEMS

SU-8 has been primarily used for structural elements and microfludics components in MEMS. Microsystems for biological applications require immobilization of biomolecules on the MEMS structures. In order to functionalize SU-8 for such purposes, the surface needs to be modified. In this paper, we report a novel dry method of surface modification of SU-8 which is compatible with standard microfabrication techniques. The surface obtained by spin coating SU-8 (2002) on silicon wafer was modified by grafting amine groups using pyrolytic dissociation of ammonia in a hotwire CVD setup. To demonstrate the presence of amine groups on modified SU-8 surface, the surface characteristic after modification was assessed using Fourier transform infrared spectroscopy. The change in SU-8 surface morphology before and after surface modification was investigated using atomic force microscopy. To show the utility of this process for application in Bio-MEMS, SU-8 microcantilevers were fabricated and subjected to the same surface modification protocol. Following this, the cantilevers were incubated first in a suspension of human immunoglobulin (HIgG) and then in FITC tagged goat anti-human IgG in order to demonstrate the utility of the surface modification performed. The efficacy of the process was assessed by observing the cantilevers under a fluorescence microscope.     

Targeting biomolecules with reversible covalent chemistry

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Glucose oxidase immobilization on a novel cellulose acetate-polymethylmethacrylate membrane

Glucose oxidase (GOD) was immobilized on cellulose acetate-polymethylmethacrylate (CA-PMMA) membrane. The immobilized GOD showed better performance as compared to the free enzyme in terms of thermal stability retaining 46% of the original activity at 70 degrees C where the original activity corresponded to that obtained at 20 degrees C. FT-IR and SEM were employed to study the membrane morphology and structure after treatment at 70 degrees C. The pH profile of the immobilized and the free enzyme was found to be similar. A 2.4-fold increase in Km value was observed after immobilization whereas Vmax value was lower for the immobilized GOD. Immobilized glucose oxidase showed improved operational stability by maintaining 33% of the initial activity after 35 cycles of repeated use and was found to retain 94% of activity after 1 month storage period. Improved resistance against urea denaturation was achieved and the immobilized glucose oxidase retained 50% of the activity without urea in the presence of 5M urea whereas free enzyme retained only 8% activity.     

A designed protein for the specific and covalent heteroconjugation of biomolecules

Bioconjugations often rely on adaptor molecules to cross-link different biomolecules. In this work, we introduce the molecular adaptor covalin, which is a protein chimera of two self-labeling proteins with nonoverlapping substrate specificity. Covalin permits a selective and covalent heteroconjugation of biomolecules displaying appropriate functional groups. Examples for the use of covalin include the specific heteroconjugation of a reporter enzyme to an antibody and of molecular probes to the surface of living cells. The efficiency and specificity of covalin-based bioconjugations together with the availability of a large variety of substrates create immediate and ubiquitous applications for covalin in bioconjugate chemistry.     

Carboxylic acid-modified polyethylene: a novel support for the covalent immobilization of polypeptides for C-terminal sequencing.

We have developed a method for the covalent immobilization of peptides, for the purpose of C-terminal sequencing, to a novel solid support, carboxylic acid-modified polyethylene (PE-COOH) film. The peptides are attached by coupling the N-terminal amino group to the activated carboxyl groups of the film. Reagents for carboxyl group activation, including 1,3-dicyclohexylcarbodiimide (DCC), 1,1'-carbonyldiimidazole (CDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1,3-diisopropylcarbodiimide (DICD) were compared. The best yields were obtained with DCC for a variety of tested peptides and averaged approximately 50%. The covalent attachment at pH 6.7 of peptides was shown to occur predominantly thorough the alpha-amino group for the peptide, SIGSLAK, which after attachment to the PE-COOH support permitted the C-terminal lysine residue to be sequenced in good yield, indicating that the epsilon-amino group of lysine is not covalently attached. This support offers a number of advantages over other solid supports, such as silica and polyvinylidene difluoride, for C-terminal sequencing including (1) stability to base and the high temperatures (65 degrees C) employed for C-terminal sequencing, (2) wettability with both aqueous and organic solvents, (3) a high capacity (1.6 nmol/mm2) for covalent coupling of polypeptides, and (4) easy divisibility into 1 x 5-mm pieces for use in our continuous flow reactor (CFR), which is also used for automated N-terminal sequencing (Shively, J.E., Miller, P., & Ronk, M., 1987, Anal. Biochem. 163, 517-529). Automated C-terminal sequencing on these supports is described in the companion paper (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68-80).     

Reversible covalent enzyme immobilization methods for reuse of carriers

Reversible immobilization techniques which allow for multiple use of the carrier are relevant for applications, such as enzymatic microreactors, biosensors with specific setups and for expensive carriers such as superparamagnetic particles. The activity of immobilized enzymes reduces with time, so that the introduction of fresh immobilized enzyme becomes necessary. Thus, methods for reversible immobilization and multiple carrier reuse can help to reduce purchase costs and facilitate reactor construction. In this work, we present a method that makes use of the reduction and oxidation of cystamine, a cleavable linker with disulfide bond and amine functionality. For a proof of principle, α-chymotrypsin was immobilized on polyethylene glycol with terminal epoxy groups using cystamine as a crosslinker. The enzyme was highly active and could be used in repeated cycles. After the enzymatic reaction was demonstrated, α-chymotrypsin was cleaved off the particle by reducing agents. The resulting thiols on the particle surface were oxidized to disulfides by means of cysteamine, the reduction product of cystamine. This way, an almost complete oxidation of surface thiols with cysteamine was possible, restoring amine functionalization for further reactions. Reduction and oxidation were repeated several times without a decrease in the extent of amine coupling. Finally, immobilization of α-chymotrypsin could be repeated with results comparable to first run.     

A new heterobifunctional reagent for immobilization of biomolecules on glass surface

Synthesis of a new heterobifunctional reagent, [N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine] (NTMTA) is described for the immobilization of a variety of biomolecules on glass surface. Its triethoxysilyl group reacts with glass surface and trifluoroethanesulfonate ester structure reacts selectively with aminoalkyl/mercaptoalkyl function in biomolecules. The immobilization can be achieved by two ways involving two steps. The first route involves the reaction of NTMTA with glass beads followed by attachment of aminoalkyl- or mercaptoalkylated biomolecules. The second one involves the reaction of biomolecules, viz., oligonucleotides, proteins, etc., with NTMTA via their aminoalkyl or mercaptoalkyl functions to form a biomolecule conjugate, which is then reacted with glass beads (unmodified) to complete immobilization process. This has been demonstrated by successful immobilization of 5'-mercaptoalkyl- or aminoalkylated oligonucleotides and some commonly used enzymes on glass beads using NTMTA reagent.     

Light-activated immobilization of biomolecules to agarose hydrogels for controlled cellular response

We describe a new method of synthesizing photolabile hydrogel materials for convenient photoimmobilization of biomolecules on surfaces or in 3-D matrixes. Dissolved agarose was modified with photolabile S-(2-nitrobenzyl)cysteine (S-NBC) via 1,1'-carbonyldiimidazole (CDI) activation of primary hydroxyl groups. S-NBC-modified agarose remained soluble and gelable with up to 5% S-NBC substitution, yet gelation was slower and the elastic modulus of the resulting gel was lower than those of unmodified agarose. Irradiating S-NBC-grafted agarose resulted in the loss of the protecting 2-nitrobenzyl groups, thereby exposing free sulfhydryl groups for biomolecular coupling. When appropriately activated with sulfhydryl-reactive groups, either peptides or proteins were effectively immobilized to the photoirradiated hydrogel matrixes, with the irradiation energy dose (i.e., irradiation time) used to control the amount of biomolecule immobilization. When the GRGDS peptide was immobilized on agarose, it was shown to be cell-adhesive and to promote neurite outgrowth from primary, embryonic chick dorsal root ganglion neurons. The immobilized GRGDS surface ligand concentration affected the cellular response: neurite length and density increased with GRGDS surface concentration at low adhesion ligand concentration and then plateaued at higher GRGDS concentration. Grafting 2-nitrobenzyl-protected compounds to hydrogel materials is useful for creating new photolabile hydrogel substrates for light-activated functional group generation and biomolecular immobilization.     

Reactive thin polymer films as platforms for the immobilization of biomolecules

Spin-coated thin films of poly(N-hydroxysuccinimidyl methacrylate) (PNHSMA) on oxidized silicon and gold surfaces were investigated as reactive layers for obtaining platforms for biomolecule immobilization with high molecular loading. The surface reactivity of PNHSMA films in coupling reactions with various primary amines, including amine-terminated poly(ethylene glycol) (PEG-NH2) and fluoresceinamine, was determined by Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), fluorescence microscopy, and ellipsometry measurements, respectively. The rate constants of PEG-NH2 attachment on the PNHSMA films were found to be significantly increased compared to the coupling on self-assembled monolayers (SAMs) of 11,11'-dithiobis(N-hydroxysuccinimidylundecanoate) (NHS-C10) on gold under the same conditions. More significantly, the PEG loading observed was about 3 times higher for the polymer thin films. These data indicate that the coupling reactions are not limited to the very surface of the polymer films, but proceed into the near-surface regions of the films. PNHSMA films were shown to be stable in contact with aqueous buffer; the swelling analysis, as performed by atomic force microscopy (AFM), indicated a film thickness independent swelling of approximately 2 nm. An increased loading was also observed by surface plasmon resonance for the covalent immobilization of amino-functionalized probe DNA. Hybridization of fluorescently labeled target DNA was successfully detected by fluorescence microscopy and surface plasmon resonance enhanced fluorescence spectroscopy (SPFS), thereby demonstrating that thin films of PNHSMA comprise an attractive and simple platform for the immobilization of biomolecules with high densities.     

Directed covalent immobilization of fluorescently labeled cytokines

Cytokines are important mediators coordinating inflammation and wound healing in response to tissue damage and infection. Therefore, immobilization of cytokines on the surface of biomaterials is a promising approach to improve biocompatibility. Soluble cytokines signal through receptors on the cell surface leading to cell differentiation, proliferation, or other effector functions. Random immobilization of cytokines on surfaces will result in a large fraction of inactive protein due to impaired cytokine--receptor interaction. We developed a strategy that combined (i) directed covalent coupling of cytokines, (ii) quantification of coupling efficiency through fluorescence detection, and (iii) a reliable protease cleavage assay to control orientation of coupling. For this purpose, fusion proteins of the SNAP-tag followed by an enterokinase recognition site, yellow fluorescent protein (YFP), and the cytokine of interest being either interleukin-6 (IL-6) or oncostatin M (OSM) were generated. The SNAP-tag is a derivative of O(6)-alkylguanine-DNA alkyltransferase that couples itself covalently to benzylguanine. Bioactivities of the SNAP-YFP-cytokines were shown to be comparable with the nontagged cytokines. Efficient coupling of SNAP-YFP-cytokines to benzylguanine-modified beads was demonstrated by flow cytometry. The fact that enterokinase treatment released most of the fluorescence from the beads is indicative for directed coupling and only marginal adsorptive binding. Cellular responses to SNAP-YFP-cytokine beads were analyzed in cellular lysates and by confocal microscopy indicating that the directionally immobilized cytokines are fully signaling competent with respect to the activation of ERK and STAT3. The strategy presented here is generally applicable for the directed covalent immobilization of fluorescently labeled proteins including the convenient and reliable control of coupling efficiency and orientation.     

Selective patterning and immobilization of biomolecules within precisely-defined micro-reservoirs

Herein, we present the fabrication of well-defined micro-reservoirs and a simple strategy to immobilize biomolecules selectively inside the reservoirs. The micro-reservoirs are fabricated using a photocurable prepolymer, which enables the formation of concrete structures with high-fidelity, so that the reservoirs are spatially-segregated from each other by rigid physical barriers. For the directed binding of the protein, two steps are involved. First, poly(ethylene glycol) (PEG) is contact-printed on those areas where the protein binding is not desired, and next, protein binding is promoted where desired via carbodiimide chemistry. Fluorescein-tagged albumin is successfully immobilized inside the micro-reservoirs and microchannel arrays with high sensitivity, regardless of the sizes of the reservoirs and channels. The proposed system can be used for constructing multi-functional biosensors by immobilizing individual bioorganisms specifically in each micro-reservoir or microchannel.     

Comparison of physical and covalent immobilization of lipase from Candida antarctica on polyamine microspheres of alkylamine matrix

Abstract

Polyamine microspheres (PA-M) prepared using polyethyleneimine as matrix were used for the immobilization of Candida antarctica lipase. The isoelectric point of PA-M is 10.6, and the hydrophobicity of PA-M was indicated using naphthalene. Optimization of conditions showed that the maximal loading of lipase on PA-M reached 230.2 mg g^{? 1} at pH 9.0 and 35°C. An increased buffer concentration had no effect on the activity of lipase but decreased the amount of lipase adsorbed. Simulation with Langmuir and Freundlich isotherms demonstrated that the adsorption of lipase on PA-M was thermodynamically favorable. Covalent crosslinking of the lipase adsorbed extended the pH range and increased the optimal temperature of the lipase activity. The physically adsorbed lipase (P-lipase) and the covalently immobilized derivative (C-lipase) retained more than 75% and 85% of their initial activity, respectively, after 10 cycles of usage. The half-lives of P-lipase and C-lipase at 50°C were 15.70 and 27.67 times higher than that of the free enzyme, respectively. Compared to P-lipase, covalent immobilization obviously reduced the catalytic efficiency and activation energy of the enzyme.     

Controlled three-dimensional immobilization of biomolecules on chemically patterned surfaces

We used electron-beam lithography to fabricate chemical nanostructures, i.e. amino groups in aromatic self-assembled monolayers (SAMs) on gold surfaces. The amino groups are utilized as reactive species for mild covalent attachment of fluorescently labeled proteins. Since non-radiative energy transfer results in strong quenching of fluorescent dyes in the vicinity of the metal surfaces, different labeling strategies were investigated. Spacers of varying length were introduced between the gold surface and the fluorescently labeled proteins. First, streptavidin was directly coupled to the amino groups of the SAMs via a glutaraldehyde linker and fluorescently labeled biotin (X-Biotin) was added, resulting in a distance of approximately 2 nm between the dyes and the surface. Scanning confocal fluorescence images show that efficient energy transfer from the dye to the surface occurs, which is reflected in poor signal-to-background (S/B) ratios of approximately 1. Coupling of a second streptavidin layer increases the S/B-ratio only slightly to approximately 2. The S/B-ratio of the fluorescence signals could be further increased to approximately 4 by coupling of an additional fluorescently labeled antibody layer. Finally, we introduced tetraethylenepentamine as functional spacer molecule to diminish fluorescence quenching by the surface. We demonstrate that the use of this spacer in combination with multiple antibody layers enables the controlled fabrication of highly fluorescent three-dimensional nanostructures with S/B-ratios of >20. The presented technique might be used advantageously for the controlled three-dimensional immobilization of single protein or DNA molecules and the well-defined assembly of protein complexes.     

Palladium hexacyanoferrate hydrogel as a novel and simple enzyme immobilization matrix for amperometric biosensors

An amperometric glucose biosensor with glucose oxidase (GOx) immobilized into palladium hexacyanoferrate (PdHCF) hydrogel has been prepared and evaluated. The sensor was based on a two-layer configuration with biocatalytic and electrocatalytic layers separately deposited onto the electrode. To reduce the overpotential for reduction of hydrogen peroxide liberated in the enzyme catalyzed oxidation of glucose, an inner thin layer of nickel hexacyanoferrate (NiHCF) electrodeposited onto the surface of graphite electrode was used as an electrocatalyst. As an outer layer, the hydrogel of palladium hexacyanoferrate with entrapped glucose oxidase was used. Under optimal operating conditions (pH 5.0 and E = -0.075 V versus calomel (3.0 M KCl) reference electrode), sensor showed high sensitivity to glucose (0.3-1.0 microA/mM) and a response time of less than 30s. The linear response to glucose was obtained in the concentration range between 0.05 and 1.0 mM in batch analysis mode and 0-7.0 mM in FIA. During the 32 days testing period, no significant decrease in the sensor sensitivity was observed. The sensor was applied for the determination of glucose concentration in fruit juice and yoghurt drink, and the results obtained showed good correlation with results obtained by reference spectrophotometric enzyme method.     

Plasma modified surfaces for covalent immobilization of functional biomolecules in the absence of chemical linkers: towards better biosensors and a new generation of medical implants

Plasma modification and plasma polymer deposition are valuable technologies for the preparation of surfaces for the covalent binding of biomolecules for applications such as biosensors, medical prostheses, and diagnostic devices as well as surfaces for enzyme-mediated reactions. Covalency is conveniently tested by the ability of the surface to retain the attached molecules after vigorous washing with sodium dodecyl sulphate (SDS). Covalency is indicated if the fraction of protein retained lies above the curve characteristic of physisorption. Confidence in covalency is strengthened when the washing protocol is aggressive enough to remove all adsorbed protein from a control significantly more hydrophobic than the test surface. The use of linker chemistry to space the molecules from the surface is in some cases beneficial. However, the use of linker chemistry is not necessary to retain molecular function for long periods when the polymer surface is modified by energetic bombardment. The energetic bombardment retains hydrophilicity of the surface by crosslinking the subsurface, and this appears to facilitate retention of protein function. Energetic bombardment also increases the functional life of molecules immobilized and then freeze dried on plasma-modified surfaces. Analysis of the surfaces shows that the covalent binding mechanism is related to the presence of free radicals on the surface and in the subsurface regions. The unpaired electrons associated with the radicals appear to be mobile within the modified region and can diffuse to the surface to take part in binding interactions. Proactive implantable devices can make use of these principles of covalent attachment by seeding the surface of an implant with a biomolecule that elicits the desired interaction with cells and prevents undesirable responses.     

Green coconut fiber: a novel carrier for the immobilization of commercial laccase by covalent attachment for textile dyes decolourization

Commercial laccase formulation was immobilized on modified green coconut fiber silanized with 3-glycidoxypropyltrimethoxysilane, aiming to achieve a cheap and effective biocatalyst. Two different strategies were followed: one point (pH 7.0) and multipoint (pH 10.0) covalent attachment. The influence of immobilization time on enzymatic activity and the final reduction with sodium borohydride were evaluated. The highest activities were achieved after 2?h of contact time in all situations. Commercial laccase immobilized at pH 7.0 was found to have higher activity and higher affinity to the substrate. However, the immobilization by multipoint covalent attachment improved the biocatalyst thermal stability at 50?°C, when compared to soluble enzyme and to the immobilized enzyme at pH 7.0. The Schiff's bases reduction by sodium borohydride, in spite of causing a decrease in enzyme activity, showed to contribute to the increase of operational stability through bonds stabilization. Finally, these immobilized enzymes showed high efficiency in the continuous decolourization of reactive textile dyes. In the first cycle, the decolourization is mainly due to dyes adsorption on the support. However, when working in successive cycles, the adsorption capacity of the support decreases (saturation) and the enzymatic action increases, indicating the applicability of this biocatalyst for textile wastewater treatment.     

A novel and simple biomolecules immobilization method: electro-deposition ZrO2 doped with HRP for fabrication of hydrogen peroxide biosensor

For the first time, a very novel and simple immobilization method for fabrication of hydrogen peroxide biosensor was reported in this paper. The biocompatible composite HRP-ZrO(2) thin films were synthesized on gold electrode surface based on electro-deposition zirconia doped with horseradish peroxidase (HRP) by cyclic voltammetry scanning in KCl solution containing ZrO(2) and HRP. The fabricated process of biosensor was characterized by electrochemical impedance spectroscopy (EIS) and the surface topography of the prepared films was imaged by atomic force microscope (AFM). The HRP in HRP-ZrO(2) thin films kept its bioactivity and exhibited excellent electrocatalytical response to the reduction of H(2)O(2). Experimental conditions influencing the biosensor performance such as pH, potential were optimized. The resulting biosensor (HRP-ZrO(2)/Au electrode) showed a linear response to H(2)O(2) over a concentration range from 0.02 to 9.45mM with a detection limit of 2muM based on a signal-to-noise ratio of 3 under optimized conditions. The apparent Michaelis-Menten constant (K(M)(app)) was evaluated to be 8.01mM, which indicated the HRP in HRP-ZrO(2) thin films kept its native bioactivity and had high affinity for H(2)O(2). Moreover, the proposed biosensor showed high sensitivity, good reproducibility and long-term stability. What is more, this immobilization methodology widened biosensor application in biomolecules immobilization and could further develop for other protein and biomolecules immobilization.