Optimization of protease immobilization by covalent binding using glutaraldehyde

Immobilization of a protease, Flavourzyme, by covalent binding on various carriers was investigated. Lewatit R258-K, activated with glutaraldehyde, was selected among the tested carriers, because of the highest immobilized enzyme activity. The optimization of activation and immobilization conditions was performed to obtain high recovery yield. The activity recovery decreased with increasing carrier loading over an optimal value, indicating the inactivation of enzymes by their reaction with uncoupled aldehyde groups of carriers. The buffer concentrations for carrier activation and enzyme immobilization were optimally selected as 500 and 50 mM, respectively. With increasing enzyme loading, the immobilized enzyme activity increased, but activity recovery decreased. Immobilization with a highly concentrated enzyme solution was advantageous for both the immobilized enzyme activity and activity recovery. Consequently, the optimum enzyme and carrier loadings for the immobilization of Flavourzyme were determined as 1.8 mg enzyme/mL and 0.6 g resin/mL, respectively.     

Optimization of glutaraldehyde activation of a support for enzyme immobilization

The influence of several parameters (support porosity, glutaraldehyde concentration, time of action, pH) on the activation reaction of an amine porous silica (Spherosil) with glutaraldehyde has been studied. Glutaraldehyde binding onto the support was followed by measuring the carbon content of the activated silica.We established comparisons between the quantity of glutaraldehyde retained on the support after activation and the capacity of the activated silica to bind trypsin. We have defined the optimal conditions for Spherosil activation and prepared derivatives with high enzymatic activity.Our results are in agreement with a reaction mechanism with glutaraldehyde in a polymeric form resulting from aldol condensation, rather than in a monomeric form.     

Biomolecule immobilization techniques for bioactive paper fabrication

Research into paper-based sensors or functional materials that can perform analytical functions with active recognition capabilities is rapidly expanding, and significant research effort has been made into the design and fabrication of bioactive paper at the biosensor level to detect potential health hazards. A key step in the fabrication of bioactive paper is the design of the experimental and operational procedures for the immobilization of biomolecules such as antibodies, enzymes, phages, cells, proteins, synthetic polymers and DNA aptamers on a suitably prepared paper membrane. The immobilization methods are concisely categorized into physical absorption, bioactive ink entrapment, bioaffinity attachment and covalent chemical bonding immobilization. Each method has individual immobilization characteristics. Although every biomolecule–paper combination has to be optimized before use, the bioactive ink entrapment method is the most commonly used approach owing to its general applicability and biocompatibility. Currently, there are four common applications of bioactive paper: (1) paper-based bioassay or paper-based analytical devices for sample conditioning; (2) counterfeiting and countertempering in the packaging and construction industries; (3) pathogen detection for food and water quality monitoring; and (4) deactivation of pathogenic bacteria using antimicrobial paper. This article reviews and compares the different biomolecule immobilization techniques and discusses current trends. Current, emerging and future applications of bioactive paper are also discussed.     

The chemistry of enzyme and protein immobilization with glutaraldehyde

Immobilization of proteins to solid matrices has been performed for the last thirty years and has provided numerous examples of successful preparations with use in enzyme reactors, sensor preparation and immunodiagnostics. Among the arsenal of coupling reagents and procedures, glutaraldehyde plays a critical role due to its reliability and ease of use. It displays a complex chemistry that is transparent to most practitioners of immobilization. In this article we detail the structure and reactivity of glutaraldehyde protein immobilization.     

Amperometric hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase by carbon-coated iron nanoparticles in combination with chitosan and cross-linking of glutaraldehyde

Magnetic carbon-coated iron nanoparticles were used to immobilize horseradish peroxidase on the surface of a polythionine modified glassy carbon electrode in combination with chitosan and cross-linking of glutaraldehyde. The electrochemical character of this enzyme electrode and its electrocatalytic reduction to H₂O₂ were studied by cyclic voltammetry. The effects of the common experimental variables were investigated. Under the optimum conditions, this method could be successfully used for the amperometric determination of H₂O₂ in a wide concentration range of 9.6 μM to 3.16 mM with a detection limit of 3.6 μM (S/N=3). Besides, the biosensor also exhibited good selectivity, stability and reproducibility.

     

Site-selective immobilization of gold nanoparticles functionalized with DNA oligomers

The organization of metal and semiconductor nanoparticles to form micro- and nanostructured assemblies is currently of tremendous interest. This communication reports on the utilization of DNA molecules as positioning elements for generating microstructured surface architecture from gold nanoparticles. Citrate-passivated 40 nm gold colloids were modified by chemisorptive coupling with a 5′-thiol-derivatized DNA oligomer. The nucleic acid was used as a molecular handle for the specific immobilization on solid supports, previously functionalized with capture DNA oligomers, complementary to the nanoparticle-bound DNA. As a consequence of the enormous specificity of nucleic acid hybridization, the DNA-directed immobilization (DDI) allows, to site-specifically target the hybrid nanoparticles to microlocations which contain the complementary oligomers. The site-selectivity of the surface adsorption is demonstrated by immobilizing the gold colloids on a DNA microarray on a glass cover slide. Moreover, scanning force microscopy (SFM) analysis, used to characterize the intermediate steps of the DDI on a gold substrate, provided initial insights into the specificity and efficiency of this technique. The application of the DDI to fabricate complex colloidal micro- and nanostructures is anticipated. Received: 26 July 2000/Accepted: 5 October 2000     

Cross-linked enzyme aggregates: a simple and effective method for the immobilization of penicillin acylase

[reaction--see text] Penicillin G acylase (penicillin amidohydrolase, E.C. 3.5.1.11) was immobilized in a simple and effective way by physical aggregation of the enzyme, using a precipitant, followed by chemical cross-linking to form insoluble cross-linked enzyme aggregates (CLEAs). These had the same activity in the synthesis of ampicillin as cross-linked crystals of the same enzyme, but the accompanying hydrolysis of the side-chain donor was much less. Penicillin G acylase CLEAs also catalyzed the synthesis of ampicillin in a broad range of organic solvents.     

Hydrogen-bonding interactions in acetonitrile oligomers using density functional theory method

Hydrogen-bonding interaction in acetonitrile oligomers is studied using density functional theory method. Two types of hydrogen-bonded oligomers are considered viz. cyclic and ladder. Different levels are used to optimize the geometry of acetonitrile monomer and found that at B3LYP/aug-cc-pvtz level the geometrical parameters and vibrational frequencies are in agreement with the experimental determinations. The BSSE corrected total energies of acetonitrile oligomers show that the cyclic structures are more stable than the ladder and the hydrogen bonds in former are stronger than those in the latter. Many-body analysis approach was used to study the nature of interactions between different molecules in these oligomers. It is found that the contribution from many-body energies to the binding energy of a complex is different in cyclic and ladder structures. An increase and decrease in the energy per hydrogen bond with cluster size for the cyclic and ladder structures, respectively, indicates the positive and negative hydrogen-bond cooperativity, respectively.     

Glucose sensor using a phospholipid polymer-based enzyme immobilization method

An electroenzymatic glucose sensor based on a simple enzyme immobilization technique was constructed and tested. The glucose sensor measures glucose concentrations as changes of oxygen concentrations induced by enzymatic reactions. The immobilizing procedure was developed with the purpose of producing wearable biosensors for clinical use. Two types of biocompatible polymers, 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymerized with dodecyl methacrylate (PMD) and MPC copolymerized with 2-ethylhexyl methacrylate, were compared as a sensitive membrane of biosensors. The PMD enzyme membrane had a better response time. Linearity, reproducibility, effect of the concentrations of immobilized enzyme and drifts of sensor characteristics in long-term tests were also investigated. The linear characteristics were confirmed with glucose concentration from 0.01 to 2.00 mmol/l, with a coefficient of determination of 0.9999. The average output current for 1 mmol/l and the standard deviation were 0.992 and 0.0283 muA. Significant changes in the sensor's characteristics were not observed for 2 weeks when it was kept in a refrigerator at 4 degrees C. Because of the simple procedure, the enzyme immobilization method is not only useful for wearable devices but also other devices such as micro total analysis systems.     

Use of titaniumetc. Species for the immobilization of bioactive compounds — Enzymes

Conclusions From the results reported in this review on immobilization of enzymes and biomolecules by metal-link/chelation processes several general conclusions can be drawn.The original metal-link process is a very simple immobilization procedure which led to very high active preparations, but with relatively low operational stabilities, mainly with macromolecular substrates (starch, casein).Difficult chemical types of carriers can be activated for enzyme immobilization, the organic supports being more easily activated than the inorganic ones. With these last type of support thetransition metal salt activation is formed by the hydrophilicity (free hydroxyl groups) of the support surface. Nevertheless, when free hydroxyl groups are not available on the support surface, the immobilization matrix can be activated byin situ precipitation of hydrous metal oxide on its surface.The use of hydrous metal oxidesper se as internal supports in the easiest route to immobilize biomolecules. However, its major drawback is that they do not possess physical structure to be used successfully in continuous reactions, thus it might be combined with an inert material.Author to whom all correspondence should be directed.     

Novel immobilization method of enzymes using a hydrophilic polymer support

A novel immobilization of an enzyme with a hydrophilic polymer support in organic solvents has been developed utilizing the "polymer-incarcerated (PI) method", which has been used to immobilize metal catalysts; the kinetic resolution of secondary alcohols was found to proceed more smoothly using immobilized lipases (CALB) than free lipases.     

Novel method for the immobilization of nucleotides

[reaction: see text] A novel method for the immobilization of nucleotides has been developed. The strategy employs a highly reactive pyrrolidinium phosphoramidate zwitterion intermediate that undergoes nucleophilic attack by long-chain alkylamine-controlled pore glass (LCAA-CPG) to generate an immobilized nucleotide. Quantification of nucleotide loading was accomplished by acidic hydrolysis of the P-N bond and subsequent HPLC analysis of TMP in the presence of an internal standard. Typical nucleotide loadings of 51-59 micromol/g of support were observed.     

FTIR investigation of cross-linking and isomerization reactions of acetylene-terminated polyimide and polyisoimide oligomers

The interrelationship between the cross-linking and isoimide-imide isomerization reactions of oligomeric isoimides and imides was investigated using FTIR spectroscopy. It was found that cross-linking of the acetylene units of Thermid IP-600 retarded the local rearrangement of the isoimide to imide conversion of Thermid IP-600 compared with an aniline-terminated analog, decreasing the rate constant by a factor of about 2. The increased flexibility of both the isoimide (Thermid IP-600) and imide (Thermid FA-700) oligomers increased the cross-linking rate of the acetylene end groups by a factor of 4 and 2, respectively, over that of the more rigid imide oligomer (MC-600). With increasing conversion of the isoimide to imide linkages in Thermid IP-600, the kinetic parameters, E_α and in A, for the cross-linking reaction of this oligomer became the same as those for the oligomeric imide, Thermid MC-600. © 1994 John Wiley & Sons, Inc.     

Development of immobilization technique for liver microsomes

In the present report, physically adsorbed rat liver microsomes were used in order to optimize the immobilization of membrane proteins on solid surfaces for use in biosensing and microreactor applications. Physical adsorption was used to form thin films on solid supports (gold, mica, macroporous aluminum oxide membrane). The characterization of the films was performed by surface plasmon resonance (SPR), atomic force microscopy (AFM) and environmental scanning electron microscopy (ESEM). Commercially available macroporous aluminium oxide membranes with a high surface area, allow the retention of a high amount of microsomal membranes in the form of a thin film. Microsomal film functionality was tested by monitoring the activities of several enzymes of phases I and II. Microsomal modified supports can be re-utilized for the same or different substrate after washing with appropriate buffer.     

Novel method for catalyst immobilization using an ionic polymer: a case study using recyclable ytterbium triflate

A series of ionic polymers prepared by quarternization of cross-linked poly(4-vinylpyridine/styrene) (P/S) resins with several alkylating agents, including short-length PEG mesylate were used as polymeric supports to immobilize Yb(OTf)₃. The efficacy of the polymer-bound catalyst was examined in a Mannich-type reaction.     

Development of cost effective method for production of 64Cu from natNi

A ^natNi foil was used for the production of ⁶⁴Cu via ⁶⁴Ni(p,n)⁶⁴Cu nuclear reaction when the necessary investment for target material (350 mg) is 50 times less using the ^natNi instead of ⁶⁴Ni. The produced 32.2 ± 1.8 MBq of “no carrier added” ⁶⁴Cu is sufficient for 10 mice trials on small animal PET. The radionuclide contamination was <13 ± 12 kBq for ⁵⁵Co and 4 ± 2 kBq for ⁵⁷Ni comparing to minimum detectable activity and only 52 ± 2 kBq of ⁶¹Cu was in ⁶⁴Cu due to the modified ion exchange separation. The concentration of Fe(III) was maintained under 1.7 ppm by precipitation and filtering of Fe(OH)₃ due to the chemical purity was required.     

Trypsin immobilization in ordered porous polymer membranes for effective protein digestion

Fast and effective protein digestion is a vital process for mass spectrometry (MS) based protein analysis. This study introduces a porous polymer membrane enzyme reactor (PPMER) coupled to nanoflow liquid chromatography-tandem MS (nLC-ESI-MS/MS) for on-line digestion and analysis of proteins. Poly (styrene-co-maleic anhydride) (PS-co-MAn) was fabricated by the breath figure method to make a porous polymer membrane in which the MAn group was covalently bound to enzyme. Based on this strategy, microscale PPMER (μPPMER) was constructed for on-line connection with the nLC-ESI-MS/MS system. Its capability for enzymatic digestion with bovine serum albumin (BSA) was evaluated with varied digestion periods. The on-line proteolysis of BSA and subsequent analysis with μPPMER-nLC-ESI-MS/MS revealed that peptide sequence coverage increased from 10.3% (digestion time 10 min) to 89.1% (digestion time 30 min). μPPMER can efficiently digest proteins due to the microscopic confinement effect, showing its potential application in fast protein identification and protease immobilization. Applications of on-line digestion using μPPMER with human plasma and urinary proteome samples showed that the developed on-line method yielded equivalent or better performance in protein coverage and identified more membrane proteins than the in-solution method. This may be due to easy accommodation of hydrophobic membrane proteins within membrane pores.     

Synthesis of a glutaraldehyde derivative of calix[4]arene as a cross-linker reagent for lipase immobilization

This article describes the synthesis of a new calix[4]arene 1,3-distal glutaraldehyde derivative 4 as a cross-linker-reagent for immobilization of Candida rugosa lipase (CRL). p-tert-Butylcalix[4]arene 1,3-distal diaminoalkyl derivative (3) synthesized via aminolysis reaction of 5,11,17,23-tert-butyl-25,27-ethoxycarbonylmethoxy-26,28-hydroxycalix[4]arene (2) with 1,8-diaminooctane. Compound 3 was converted to its aldehyde derivative (4) by the treatment with glutaraldehyde solution. 4 was used in lipase immobilization in order to see the role of calix[4]arene binding site on the lipase activity and stability. It was observed that the immobilized lipase activity was maintained at levels exceeding 95% of its original activity after 40 min.     

Impedimetric immunosensor using avidin-biotin for antibody immobilization

A newly developed electrochemical method--Elimination Voltammetry with Linear Scan (EVLS)--has been applied to the electrochemical study of nucleic acids (NAs) on a silver electrode. Using the linear combination of the currents measured at different scan rates, the EVLS is capable of eliminating one or two selected particular currents. It was shown that the elimination function conserving the reversible diffusion current and eliminating the charging and kinetic currents provides the significant increase of voltammetric signals of DNA. Due to the high sensitivity and resolution power, the EVLS can contribute to study behaviour of nucleic acids on the charged interface and can be applied to nucleic acid analyses and the development of DNA sensors.