Enzyme Immobilization Strategies Research Articles

Facile preparation of porous magnetic polydopamine microspheres through an inverse replication strategy for efficient enzyme immobilization.

An inverse replication method based on porous CaCO3 templates was developed to fabricate porous magnetic polymer microspheres (PMMSs) composed of biocompatible polydopamine and magnetic Fe3O4 nanoparticles. The preparation procedure involved the synthesis of Fe3O4@CaCO3 templates, infiltration and spontaneous polymerization of dopamine in template pores and finally the mild removal of templates. The particle size, the surface morphology and the pore structure (e.g., average pore size, pore volume, surface area, etc.) of porous PMMSs were facilely tailored by altering the templates. The as-prepared polydopamine microspheres PMMSs were applied to covalently immobilize YADH for catalyzing the conversion of formaldehyde to methanol. In comparison to the enzyme-conjugated PDA-coated Fe3O4 nanoparticles (PMNPs), the immobilized enzyme on porous PMMSs exhibited remarkably enhanced activity (specific activity: 162.3 U mg-1 enzyme vs. 97.6 U mg-1 enzyme; methanol yield: 95.5% vs. 57.1%; initial reaction rate: 0.15% s-1vs. 0.08% s-1), and desirable thermal/pH/recycling/storage stabilities, and particularly, easy separation from the bulk solution by an external magnetic field.

A general strategy for site-directed enzyme immobilization by using NiO nanoparticle decorated mesoporous silica.

Mesoporous materials have recently gained much attention owing to their large surface area, narrow pore size distribution, and superior pore structure. These materials have been demonstrated as excellent solid supports for immobilization of a variety of proteins and enzymes for their potential applications as biocatalysts in the chemical and pharmaceutical industries. However, the lack of efficient and reproducible methods for immobilization has limited the activity and recyclability of these biocatalysts. Furthermore, the biocatalysts are usually not robust owing to their rapid denaturation in bulk solvents. To solve these problems, we designed a novel hybrid material system, mesoporous silica immobilized with NiO nanoparticles (SBA-NiO), wherein enzyme immobilization is directed to specific sites on the pore surface of the material. This yielded the biocatalytic species with higher activity than free enzyme in solution. These biocatalytic species are recyclable with minimal loss of activity after several cycles, demonstrating an advantage over free enzymes.

Laccase biosensors based on different enzyme immobilization strategies for phenolic compounds determination

Different enzyme immobilization approaches of Trametes versicolor laccase (TvL) onto gold surfaces and their influence on the performance of the final bioanalytical platforms are described. The laccase immobilization methods include: (i) direct adsorption onto gold electrodes (TvL/Au), (ii) covalent attachment to a gold surface modified with a bifunctional reagent, 3,3'-Dithiodipropionic acid di (N-succinimidyl ester) (DTSP), and (iii) integration of the enzyme into a sol–gel 3D polymeric network derived from (3-mercaptopropyl)-trimethoxysilane (MPTS) previously formed onto a gold surface (TvL/MPTS/Au). The characterization and applicability of these biosensors are described. Characterization is performed in aqueous acetate buffer solutions using atomic force microscopy (AFM), providing valuable information concerning morphological data at the nanoscale level.The response of the three biosensing platforms developed, TvL/Au, TvL/DTSP/Au and TvL/MPTS/Au, is evaluated in the presence of hydroquinone (HQ), used as a phenolic enzymatic substrate. All systems exhibit a clear electrocatalytic activity and HQ can be amperometrically determined at −0.10V versus Ag/AgCl. However, the performance of biosensors – evaluated in terms of sensitivity, detection limit, linear response range, reproducibility and stability – depends clearly on the enzyme immobilization strategy, which allows establishing its influence on the enzyme catalytic activity.

Bioactive paper: Biomolecule immobilization methods and applications in environmental monitoring

Abstract

Plasmon-Enhanced Enzymatic Reactions 2:Optimization of Enzyme Activity by Surface Modification of Silver Island Films with Biotin-Poly (Ethylene-glycol)-Amine

Surface modification of silver island films (SIFs) was carried out with Biotin-Poly (Ethylene-glycol)-Amine (BEA), which acts as a cross-linker between the silver surface and horse radish peroxidase (HRP) enzyme for optimum plasmon-enhanced enzymatic activity. SIFs-deposited blank glass slides and SIFs-deposited 3-Aminopropyltriethoxysilane(APTES)-coated glass slides were used as our plasmonic surfaces.In this regard, three different extent of loading of SIFs were also prepared (low, medium and high) on APTES-coated glass slides. Streptavidin-linked HRP enzyme was attached to SIFs-deposited blank glass slides and SIFs-deposited APTES-coated glass slides through the well-known biotin-streptavidin interactions. The characterization of these surfaces was done using optical absorption spectroscopy. The loading of SIFs on glass slides was observed to have significant effect on the efficiency of plasmon-enhanced enzymatic activity, where an enhancement of 200% in the enzymatic activity was observed when compared to our previously used strategies for enzyme immobilization in our preceding work[1]. In addition, SIFs-deposited on APTES-coated glass slides were found to be re-usable for plasmon-enhanced enzymatic reactions unlike SIFs deposited on to blank glass slides.

Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance

AbstractEnzyme biocatalysis plays a very relevant role in the development of many chemical industries, e.g., energy, food or fine chemistry. To achieve this goal, enzyme immobilization is a usual pre‐requisite as a solution to get reusable biocatalysts and thus decrease the price of this relatively expensive compound. However, a proper immobilization technique may permit far more than to get a reusable enzyme; it may be used to improve enzyme performance by improving some enzyme limitations: enzyme purity, stability (including the possibility of enzyme reactivation), activity, specificity, selectivity, or inhibitions. Among the diverse immobilization techniques, the use of pre‐existing supports to immobilize enzymes (via covalent or physical coupling) and the immobilization without supports [enzyme crosslinked aggregates (CLEAs) or crystals (CLECs)] are the most used or promising ones. This paper intends to give the advantages and disadvantages of the different existing immobilization strategies to solve the different aforementioned enzyme limitations. Moreover, the use of nanoparticles as immobilization supports is achieving an increasing importance, as the nanoparticles versatility increases and becomes more accessible to the researchers. We will also discuss here some of the advantages and drawbacks of these non porous supports compared to conventional porous supports. Although there are no universal optimal solutions for all cases, we will try to give some advice to select the optimal strategy for each particular enzyme and process, considering the enzyme properties, nature of the process and of the substrate. In some occasions the selection will be compulsory, for example due to the nature of the substrate. In other cases the optimal biocatalyst may depend on the company requirements (e.g., volumetric activity, enzyme stability, etc).

A comparative study of enzyme immobilization strategies for multi-walled carbon nanotubeglucose biosensors

This work addresses the comparison of different strategies for improving biosensorperformance using nanomaterials. Glucose biosensors based on commonly appliedenzyme immobilization approaches, including sol–gel encapsulation approaches andglutaraldehyde cross-linking strategies, were studied in the presence and absence ofmulti-walled carbon nanotubes (MWNTs). Although direct comparison of designparameters such as linear range and sensitivity is intuitive, this comparison alone is notan accurate indicator of biosensor efficacy, due to the wide range of electrodesand nanomaterials available for use in current biosensor designs. We proposed acomparative protocol which considers both the active area available for transductionfollowing nanomaterial deposition and the sensitivity. Based on the protocol, whenno nanomaterials were involved, TEOS/GOx biosensors exhibited the highestefficacy, followed by BSA/GA/GOx and TMOS/GOx biosensors. A novel biosensorcontaining carboxylated MWNTs modified with glucose oxidase and an overlyingTMOS layer demonstrated optimum efficacy in terms of enhanced current density (18.3 ± 0.5 µA mM − 1 cm − 2), linear range (0.0037–12 mM), detection limit (3.7 µM),coefficient of variation (2%), response time (less than 8 s), and stability/selectivity/reproducibility.H2O2 response tests demonstrated that the most possible reason for the performanceenhancement was an increased enzyme loading. This design is an excellent platform forversatile biosensing applications.

In situ entrapment of urease in cryogels of poly(N‐isopropylacrylamide): An effective strategy for noncovalent immobilization of enzymes

AbstractSupermacroporous poly(N‐isopropylacrylamide) (PNIPAAm) cryogels containing urease were prepared via UV irradiation technique and hydrogen peroxide as initiator. Specifically, due to the cryostructuration phenomenon urease molecules were embedded into the dense cryogel walls. Thus, although the enzyme is physically entrapped, the system exhibited remarkable resistance against leaking due to the dense polymer network formed in the cryogel walls. The immobilized urease can catalyze the hydrolysis of urea in a broad temperature range in both batch and flow regime. The interconnected macropores assist for unhindered diffusion of the substrate and reaction products through the gel, thus, paving the way for consecutive reuse at a constant activity, in contrast to the conventional PNIPAAm hydrogel. Due to the spongy‐like morphology PNIPAAm cryogels containing urease can be exploited as highly permeable membrane for direct removal of traces of urea from continuously flowing feed solutions. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Peptide-Modified Surfaces for Enzyme Immobilization

BackgroundChemistry and particularly enzymology at surfaces is a topic of rapidly growing interest, both in terms of its role in biological systems and its application in biocatalysis. Existing protein immobilization approaches, including noncovalent or covalent attachments to solid supports, have difficulties in controlling protein orientation, reducing nonspecific absorption and preventing protein denaturation. New strategies for enzyme immobilization are needed that allow the precise control over orientation and position and thereby provide optimized activity.Methodology/Principal FindingsA method is presented for utilizing peptide ligands to immobilize enzymes on surfaces with improved enzyme activity and stability. The appropriate peptide ligands have been rapidly selected from high-density arrays and when desirable, the peptide sequences were further optimized by single-point variant screening to enhance both the affinity and activity of the bound enzyme. For proof of concept, the peptides that bound to β-galactosidase and optimized its activity were covalently attached to surfaces for the purpose of capturing target enzymes. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activity and stability, as well as controlled protein orientation.Conclusions/SignificanceA simple method for immobilizing enzymes through specific interactions with peptides anchored on surfaces has been developed. This approach will be applicable to the immobilization of a wide variety of enzymes on surfaces with optimized orientation, location and performance, and provides a potential mechanism for the patterned self-assembly of multiple enzymes on surfaces.

A DNA-based strategy for dynamic positional enzyme immobilization inside fused silica microchannels

A three-enzyme cascade reaction was successfully realized in a continuous flow microreactor. The first enzyme (Candida antarctica lipase B, also known as Pseudozyma antarctica lipase B) and the third enzyme (horseradish peroxidase) of the cascade process were immobilized in a mild non-contact manner via ssDNA-ssDNA interaction in discrete zones on the capillary wall, whereas the second enzyme (glucose oxidase) was kept in the mobile phase. The unique combined feature of patterning, possibility of loading and stripping, and modularity in a fused silica microchannel is demonstrated. By changing the distance between the two enzyme patches, the reaction time available for glucose oxidase could be independently and modularly varied. The reusability of the enzymatic microfluidic system was shown by using the hybridization and dehybridization capabilities of DNA as a tool for subsequent enzyme immobilization and removal.

Glucose Oxidase Catalyzed Self‐Assembly of Bioelectroactive Gold Nanostructures

AbstractGlucose oxidase catalyzes the formation of metallic gold particles in immediate proximity of the protein from gold (III) chloride in the absence of any other catalytic or reductive substrates. The protein‐mediated gold reduction reaction leads to size‐controllable gold particle formation and concomitant association of the enzyme in an electrically conductive metallic template. Such an enzyme immobilization strategy provides a simple and rapid method to create an intimate interface between glucose oxidase and a conductive matrix, which can be joined to an electrode surface. Model electrodes were prepared by entraining the glucose oxidase/gold particles onto carbon paper. Voltammetry of the resulting electrodes revealed stable oxidation and reduction peaks at a potential close to that of the standard value for the FAD/FADH2 cofactor of immobilized glucose oxidase. The gold electrodes exhibit catalytic activity in the presence of glucose confirming the entrapment of active glucose oxidase within the gold architecture. The resulting composite material can be successfully integrated with electrodes of various designs for biosensor and biofuel cell applications.

Development of enzyme immobilized monolith micro-reactors integrated with microfluidic electrochemical cell for the evaluation of enzyme kinetics

This paper describes a simple and efficient method for producing an on-chip enzyme immobilized monolith micro-reactor that integrates a microfluidic electrochemical cell for rapid characterization of enzymatic kinetics. The monolith was generated using a sol–gel method, followed by PEI functionalization and enzyme immobilization via electrostatic attraction between electronegative enzymes and electropositive PEI polymers. Using the proposed immobilization strategy, a glucose oxidase (GOD) immobilized monolith micro-reactor has been produced with the controllable porosity that gives better enzyme kinetics compared to previously reported devices. This can be attributed to a favourable enzyme-substrate affinity in which more than 98% of the immobilized enzyme remains in an active conformation. The kinetic studies conducted have identified that a similar value of the kcat is obtained for immobilized GOD (13.4 s−1) and GOD free in solution (14 s−1) whilst the immobilized Michaelis constant Km(app) (7.2 mM) is ~4 times lower than GOD in solution (25 mM). In addition, the immobilized GOD exhibits increased stability, retaining at least 95% of the initial activity when stored of 30 days at 4°C, compared to only 60% for GOD in solution. Furthermore, the same enzyme immobilization strategy has been used for choline oxidase immobilization and similar kinetics to choline oxidase in solution were observed, once again indicating better maintenance of the enzyme conformation provided by the proposed method.

Molecular View of the Interaction between ι-Carrageenan and a Phospholipid Film and Its Role in Enzyme Immobilization

Proteins incorporated into phospholipid Langmuir-Blodgett (LB) films are a good model system for biomembranes and enzyme immobilization studies. The specific fluidity of biomembranes, an important requisite for enzymatic activity, is naturally controlled by varying phospholipid compositions. In a model system, instead, LB film fluidity may be varied by covering the top layer with different substances able to interact simultaneously with the phospholipid and the protein to be immobilized. In this study, we immobilized a carbohydrate rich Neurospora crassa alkaline phosphatase (NCAP) in monolayers of the sodium salt of dihexadecylphosphoric acid (DHP), a synthetic phospholipid that provides very condensed Langmuir films. The binding of NCAP to DHP Langmuir-Blodgett (LB) films was mediated by the anionic polysaccharide iota-carrageenan (iota-car). Combining results from surface isotherms and the quartz crystal microbalance technique, we concluded that the polysaccharide was essential to promote the interaction between DHP and NCAP and also to increase the fluidity of the film. An estimate of DHP:iota-car ratio within the film also revealed that the polysaccharide binds to DHP LB film in an extended conformation. Furthermore, the investigation of the polysaccharide conformation at molecular level, using sum-frequency vibrational spectroscopy (SFG), indicated a preferential conformation of the carrageenan molecules with the sulfate groups oriented toward the phospholipid monolayer, and both the hydroxyl and ether groups interacting preferentially with the protein. These results demonstrate how interfacial electric fields can reorient and induce conformational changes in macromolecules, which may significantly affect intermolecular interactions at interfaces. This detailed knowledge of the interaction mechanism between the enzyme and the LB film is relevant to design strategies for enzyme immobilization when orientation and fluidity properties of the film provided by the matrix are important to improve enzymatic activity.

Enzymatic synthesis of water‐soluble conducting poly(3,4‐ethylenedioxythiophene): A simple enzyme immobilization strategy for recycling and reusing

The field of intrinsically conducting polymers has attracted high attention due to their interesting electrical and optical properties. Since their discovery in 1977, they have been investigated for many technological applications, such as organic lightweight batteries, microelectronics, optical displays, antistatic coatings, sensors, direct metallization, all-polymer actuators, and electromagnetic shielding. These highly promising materials have traditionally been synthesized by monomer oxidation in the presence of a strong oxidant and, once synthesized, they usually show high insolubility and intractability. The use of enzymes as biocatalysts for the synthesis of conducting polymers has been studied in last years as a ‘‘green synthesis process’’ alternative. Enzymes can offer environmentally benign reaction conditions, a high yield of polymerization, and higher control in regioregularity and stereochemistry, consequently resulting in soluble and processable conducting polymers. In most cases, the research was focused on the synthesis of polyaniline (PANI). According to this biocatalytic approach, the aniline is coupled together by horseradish peroxidase (HRP) or another peroxidase catalyzed oxidation in the presence of an anionic polyelectrolyte or a micellar template leading to a PANI aqueous dispersion. Moreover, in general, the expensive enzymes must be recovered and reused after the reaction for practical applications. This is the main reason behind the well established strategy of immobilization of enzymes into solid supports which was applied to the HRP enzyme. Recently, our group reported the first enzymatic polymerization of 3,4-ethylenedioxythiophene (EDOT), demonstrating that this green synthesis process can be applied to other technologically interesting polymers besides PANI. In this work, a pH of 2 and a Temperature of 4 C were established as optimum conditions for the adequate synthesis of PEDOT. And even if some previous researches reported an abrupt decrease of the enzymatic activity in acidic media (at pH 1⁄4 4, the HRP activity is around 0 after 1 h), in our case, the reaction was observed to proceed uninterruptedly throughout 16 h. Then, we proved that the enhanced enzyme activity was due to the presence of an excess of EDOT in the media (monomer droplets), where the HRP was more preferentially localized. Monomer droplets would act, consequently, as (a) enzyme protectors against deactivation as well as (b) a monomer feed to keep a constant EDOT concentration in the reaction media. In another previous contribution by the authors a biphasic catalytic system was employed as a way to recover and reuse the enzyme several times after aniline polymerization. In this case, the enzyme was encapsulated inside an ionic liquid while the aniline monomer, the H2O2 oxidant, and the template stayed in aqueous Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 47, 306–309 (2009)

Immobilization of Enzymes through One-Pot Chemical Preoxidation and Electropolymerization of Dithiols in Enzyme-Containing Aqueous Suspensions To Develop Biosensors with Improved Performance

A protocol of one-pot chemical preoxidation and electropolymerization of monomers (CPEM) in enzyme-containing aqueous suspensions (or solutions) was proposed as a universal strategy for high-activity and high-load immobilization of enzymes to construct amperometric biosensors, which was proven to be effective for the monomer of 1,4-benzenedithiol (BDT), 1,6-hexanedithiol, o-phenylenediamine, o-aminophenol or pyrrole, the preoxidant of K3Fe(CN)6 or p-benzoquinone, and the enzyme of glucose oxidase (GOx) or alkaline phosphatase (AP) to develop GOx-based glucose biosensors or AP-based disodium phenyl phosphate biosensors. As a case examined in detail, a well-dispersed aqueous suspension of the poorly soluble BDT was obtained through its dispersion assisted by ultrasonication and coexisting GOx, which was then subject to chemical preoxidation through adding K3Fe(CN)6, yielding many composites of insoluble BDT oligomers with lots of high-activity enzyme molecules entrapped. Some insoluble composites were then electrochemically codeposited with poly(1,4-benzenedithiol) on an Au electrode, yielding an enzyme film with high-load and high-activity enzyme immobilized. The glucose biosensor prepared here from the CPEM protocol showed much better performance than that from the preoxidant-free conventional electropolymerization (CEP) protocol, with a detection sensitivity increase by a factor of 32 in this case. The GOx-based and AP-based first-generation biosensors developed from the present CPEM protocol all exhibited notably improved performance compared with the analogues from the preoxidant-free CEP protocol. The electrochemical quartz crystal microbalance (EQCM) technique was used to investigate various electrode modification processes. The values of quantity and enzymatic specific activity (ESA) of the immobilized enzymes were evaluated through the EQCM and the conventional UV-vis spectrophotometric method, given that the CPEM protocol notably improved the quantity and the ESA of immobilized enzymes as compared with the preoxidant-free CEP protocol. The proposed CPEM protocol may be interesting in a number of fields, including biosensing, biocatalysis, biofuel cells, bioaffinity chromatography, and biomaterials, and the successful electropolymerization of dithiols in aqueous suspensions (two-phase electropolymerization) may open a new avenue for many monomers that are poorly soluble in neutral aqueous solutions to in situ immobilize biomolecules for bioapplications.

An efficient resolution of racemic secondary alcohols on magnetically separable biocatalyst

The combination of magnetic nanoparticles and mesoporous silica can present a strategy for enzyme immobilization. In this work, magnetic siliceous mesocellular foam functionalized with octyl groups was prepared and used as support for lipase adsorption. Almost all the active lipases in crude enzyme solution were adsorbed by this magnetically separable, hydrophobic siliceous mesocellular foam. The resolution of 1-phenylethanol acylated with vinyl acetate can be achieved in 1.5 h using the resultant magnetic biocatalyst, whereas only 30% conversion was obtained by using the free lipase under the identical reaction conditions. These results are probably due to the “interfacial adsorption” and “hyper-activation” of lipase on the hydrophobic surface of the magnetic siliceous mesocellular foam. Moreover, the biocatalyst entrapped in the nanopores of this foam can be recycled magnetically for at least seven times without significant loss of its activity and enantioselectivity.

A strategy for enzyme immobilization on layer-by-layer dendrimer–gold nanoparticle electrocatalytic membrane incorporating redox mediator

A new approach is described to produce nanostructured electrocatalytic membranes using a combination of three methods. Using the layer-by-layer technique, PAMAM dendrimers with cobalt hexacyanoferrates-modified gold nanoparticles were alternated with poly(vinylsulfonic acid) layers on ITO (indium tin oxide) electrodes. This film was used as substrate for immobilization of glucose oxidase in the presence of bovine serum albumin and glutaraldehyde as cross-linker. The modified electrode was successfully applied as a biosensor for the amperometric measurement of glucose, using glucose oxidase enzyme, at 0.0 V vs. SCE.

Nanopore-Based Proteolytic Reactor for Sensitive and Comprehensive Proteomic Analyses

Various silica-based microreactors have been designed that use enzyme immobilization to address technical concerns in proteolysis including inefficient and incomplete protein digestion. Most of current designs for proteolytic reactors can improve either protease stability or proteolysis efficiency of individual protein(s). However, the desired features such as rapid digestion, larger sequence coverage, and high sensitivity have not been achieved by a single microreactor design for broad range proteins with diverse physical properties. Here, unlike conventional enzyme immobilization strategies, we describe a novel proteolytic nanoreactor based on the unique three-dimensional nanopore structure of our newly synthesized mesoporous silica (MPS), FDU-12, which integrates substrate enrichment, "reagent-free" protein denaturation, and efficient proteolytic digestion. In our design, protein substrates were first captured by MPS nanopore structure and were concentrated from the solution. Following the pH change and applying trypsin, the denaturation and concurrent proteolysis of broad-range proteins were efficiently achieved. In minutes, many more sample peptides from the in-nanopore digestion of protein mixtures were detected by mass spectrometry, resulting in the identifications of a broad range of diverse proteins with high sequence coverage. The unique features of FDU-12 nanostructure that allow rapid, complete proteolysis and resulting enhanced sequence coverage of individual proteins were investigated by using Raman spectroscopy and comparative studies with respect to other MPSs.

Design and Characterization of Immobilized Enzymes in Microfluidic Systems

Herein we report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes. Streptavidin-conjugated alkaline phosphatase was linked to biotinylated phospholipid bilayers coated inside poly(dimethylsiloxane) microchannels and borosilicate microcapillary tubes. Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flow-controlled dilution on-chip. This allowed Lineweaver-Burk analysis to be performed from a single experiment with all the data collected simultaneously. The results revealed an enzyme turnover number of 51.1 +/- 3.2 s(-1) for this heterogeneous system. Furthermore, the same enzyme immobilization strategy was extended to demonstrate that multiple chemical reactions could be performed in sequence by immobilizing various enzymes in series. Specifically, the presence of glucose was detected by two coupled steps employing immobilized avidinD-conjugated glucose oxidase and streptavidin-conjugated horseradish peroxidase.

Strategies for the reversible immobilization of enzymes by use of biotin-bound anti-enzyme antibodies

Glucose oxidase (E.C. 1.1.3.4) is reversibly immobilized in a reactor coupled to a flow-injection analysis system using an immunological reaction. The antibody used is irreversibly immobilized on the reactor support by an avidin—biotin linkage. The bond between avidin and biotin is nearly irreversible under normal elution conditions for antibody—antigen reactions. The reactor is packed with a support on which avidin is covalently attached and a biotin-bound second antibody is passed over the reactor packing, which immobilizes this antibody. An immune complex of the enzyme, or first anti-enzyme antibody followed separately by enzyme, is introduced into the flow system, resulting in enzyme immobilization. The reactor produced can be used in the determination of 1 × 10 −11 −1 × 10 −6 mole of glucose with a sample size of 20 μl and a sample throughput of 20–30/hr. These results are comparable to or better than those obtained with glucose oxidase directly immobilized on the same support. The enzyme can be removed by elution with low-pH buffers, and the reactor regenerated by injection of the anti-enzyme antibody and the enzyme.