A coupled two-compartment model for immobilized enzyme electrodes

The effect of stirring on the transient and pseudo steady-state behavior of potentiometric and amperometric immobilized enzyme electrodes is accurately modelled by a coupled two-compartment system of nonlinear differential equations. In the first compartment, the enzyme layer, the chemistry is governed by enzyme kinetics and diffusion. This is coupled to the second compartment, the bulk solution, which is controlled only by molecular diffusion. Several results of analytical significance can be obtained by using this model: for example, it is shown how the stirring rate can be used to increase the linear dynamic range.     

Nonionic micellar liquid chromatography coupled to immobilized enzyme reactors

Immobilized enzyme reactors are used as post-column reactors to modify the detectability of analytes. An immobilized amino acid oxidase reactor was prepared and coupled to an immobilized peroxidase reactor to detect low level of amino acids by fluorescence of the homovanilic dimer produced. A cholesterol oxidase reactor was prepared to detect cholesterol and metabolites by 241 nm UV absorbance of the enone produced. The preparation of the porous glass beads with the immobilized enzymes is described. Micellar liquid chromatography is used with non-ionic micellar phases to separate the amino acids or cholesterol derivatives. It is demonstrated that the non ionic Brij 35 micellar phases are very gentle for the enzyme activity allowing the reactor activity to remain at a higher level and for a much longer time than with hydro-organic classical chromatographic mobile phases or aqueous buffers. The coupling of nonionic micellar phases with enzymatic detection gave limits of detection of 32 pmol (4.8 ng injected) of methionine and 50 pmol (19 ng injected) of 20alpha-hydroxy cholesterol. The immobilized enzyme reactors could be used continuously for a week without losing their activity. It is shown that the low efficiency obtained with micellar liquid chromatography is compensated by the possibility offered by the technique to easily adjust selectivity.     

Lysozyme refolding with immobilized GroEL column chromatography

A refolding chromatography with immobilized molecular chaperonin GroEL was studied for the reactivation of denatured-reduced lysozyme. The effect of denaturant concentration (guanidine hydrochloride, 0.1-1.5 M) in the elution buffer, the elution flow-rate, and the loading concentration and volume of the substrate protein on the reactivation yield was studied. All the operating parameters showed minor effects on the recovery yield of lysozyme mass, which remained at 90-100%, but exhibited relatively notable influences on the specific activity of the recovered lysozyme. For example, there existed an optimum denaturant concentration of about 1 M at which the highest yield of specific activity (up to 97%) was obtained. Using the immobilized GroEL column, 3 ml of the lysozyme (1 mg/ml) per batch could be refolded at an overall yield of 81%, which corresponded to a refolding productivity of 54 mg per 1 gel per h. At comparable reactivation yields (over 80%), this value of productivity was over four-times larger as that of the size-exclusion refolding chromatography reported previously (12 mg per 1 gel per h), indicating the advantage of the present system for producing a high throughput in protein refolding operations.     

Determination of volatile N-nitrosamines in irradiated fermented sausage by gas chromatography coupled to a thermal energy analyzer

Volatile N-nitrosodimethylamine (NDMA) and N-nitrosopyrrolidine (NPYR) in irradiated pepperoni and salami sausages were determined using a gas chromatography coupled to a thermal energy analyzer (GC-TEA). These fermented sausages with aerobic or vacuum packaging were irradiated at 0, 5, 10, and 20 kGy, and then stored for 4 weeks at 4 degrees C. Both NDMA and NPYR in the fermented sausage were significantly reduced by irradiation. The vacuum packaging showed significantly lower (P < 0.05) N-nitrosamine levels than that of the aerobic ones. After storage, the contents of NDMA and NPYR in the irradiated sausage were lower than those of the non-irradiated control. Results indicated that a high dose of irradiation (>10 kGy) was needed to reduce the carcinogenic N-nitrosamines in the fermented sausage during storage and the GC-TEA analysis was effective in determining the N-nitrosamines in irradiated meats even at low trace levels.     

Evaluation of porous polymer traps for analysis of four volatile N-nitrosamines using thermal desorption injection coupled with a gas chromatograph-thermal energy analyzer

Three porous polymer adsorbents, Tenax-TA, Chromosorb 102, and Chromosorb 103 were investigated as potential gas phase, trapping agents for volatile N-nitrosamines using a thermal desorption injector coupled with a gas chromatograph-thermal energy analyzer. N-Nitrosodimethylamine was used as the model N-nitrosamine for determining break-through volume, the effect of temperature on retention volume, and collection efficiency at 25 degrees C for each adsorbent. Results from these three parameters indicated that Chromosorb 103 exhibited the best adsorbent characteristics for the pre-concentration of volatile N-nitrosamines. A mixture of three dialkyl N-nitrosamines (dimethyl, diethyl, and dipropyl), and N-nitrosopyrrolidine were analyzed using a high-temperature mineral oil purge and trap procedure. Recoveries ranged from 82.2 to 102.8% at levels of 10 and 100 ng of each N-nitrosamine added.     

Repeatedly usable immobilized pH gradient in a monolithic capillary column

Glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) were used to synthesize a monolithic capillary column containing reactive epoxy groups. Glutaraldehyde was introduced and linked to the monolith after a process of amination. An aqueous solution of commercial carrier ampholytes (CAs, Ampholine) was focused in such a polymer column. The primary amino groups of CAs reacted with glutaraldehyde along the capillary. CAs were immobilized at different positions in the column according to their isoelectric points (pI), resulting in a monolithic immobilized pH gradient (M-IPG). Isoelectric focusing (IEF) was performed without CAs in such an M-IPG column. Due to the covalent attachment of the CAs this M-IPG can be repeatedly used after its preparation. Good stability, linearity, and reproducibility were obtained.     

Amperometric determination of choline with enzyme immobilized in a hybrid mesoporous membrane

Choline sensor is successfully prepared by using immobilized enzyme, i.e., choline oxidase (ChOx) within a hybrid mesoporous membrane with 12 nm pore diameter (F127M). The measurement was based on the detection of hydrogen peroxide, which is the co-product of the enzymatic choline oxidation. The determination range and the response time are 5.0-800 μM and approximately 2 min, respectively. The sensor is very stable compared to the native enzyme sensor and 85% of the initial response was maintained even after storage for 80 days. These results indicate that ChOx is successfully immobilized and well stabilized, and at the same time, enzyme reaction proceeds efficiently. Such ability of hybrid mesoporous membrane F127M suggests great promise for effective immobilization of enzyme useful for electrochemical biosensors.     

Determination of l-alanine in a flow-injection system with an immobilized enzyme reactor

A flow-injection system for the determination of l-alanine is described. Alanine dehydrogenase is immobilized on poly(vinyl alcohol) beads and used in a packed-bed enzyme reactor. The system responds linearly to injected samples (50 μl) in the concentration range 0.5–500 μM. The maximum throughput was 40 samples per hour. The immobilized enzyme reactor was stable for at least 6 weeks. Its usefulness for assay of l-alanine in serum and beverages is described.     

Spectrophotometric determination of ethanol in blood using a flow-injection system with an immobilized enzyme (alcohol dehydrogenase) reactor coupled to an on-line dialyser

The determination of ethanol in whole blood without pretreatment using spectrotometric detection with an immobilized enzyme reactor coupled to an on-line dialyser is described. Optimum working conditions are given. The effects of the injection volume and the flow-rate on the peak height using the dialyser were investigated. A good linear calibration graph over the ethanol concentration range 3–40 μg ml^?1 was observed; these concentrations correspond to 0.3?4.0 g of ethanol per 1000 g of whole blood prior to dilution. For comparison, whole blood samples were analysed by gas chromatography with direct injection. The effect of temperature on the peak height was also studied in a system without the dialyser.     

Macroporous polyacrylamide-based monolithic column with immobilized pH gradient for protein analysis

Monolithic materials were prepared in capillaries by in situ polymerization of acrylamide, glycidyl methacrylate, and N,N'-methylenebisacrylamide in the presence of 1,4-butanediol, dodecanol, and DMSO as porogens. With Ampholine attached to the surface of the porous monolith via epoxide groups, a monolithic-IPG (M-IPG) was formed and showed good mechanical and chemical stability. With such a column immobilized by Ampholine 3.5-10, IEF-MIX 3.6-9.3 was separated and good linearity was obtained. The CIEF behavior of M-IPG was evinced by comparing the current with that in the open tubular capillary. In addition, the protein mixtures excreted from lung cancer cells of rats were analyzed with such a new M-IPG column.     

Flow injection determination of choline in milk hydrolysates by an immobilized enzyme reactor coupled to a selective hydrogen peroxide amperometric sensor

A choline oxidase (ChO) immobilized enzyme reactor (IMER) prepared by glutaraldehyde coupling of the enzyme on aminopropyl modified controlled pore glass beads is described. The ChO-IMER was coupled, in a flow injection configuration system, to an interference free hydrogen peroxide amperometric sensor based on a Pt surface modified by an overoxidized polypyrrole film. The resulting analytical device responds selectively to choline and displays a sensitivity of 46.9 ± 0.2 μC mM⁻¹ and a limit of detection, calculated at a signal-to-noise ratio equal to 3, of 7 μM. Sensitivity remains constant for about 20 days and then starts to slowly deteriorate and after 2 months a 70% of the initial sensitivity was still retained. The application to choline determination in milk hydrolysates is demonstrated. Short- and long-term drift observed in the analytical response can be corrected by a bracketing technique.     

Analysis of headspace samples using off-line sorbent trapping coupled with automated thermal desorption and splitless injection onto a cryogenically cooled wide bore capillary column

Gas chromatographic equipment and procedures are described for automated splitless injection of pseudo-static headspace samples collected externally onto a sorbent trap. The GC microprocessor controls, in sequence, carrier gas backflushing of the sorbent trap for water removal, splitless thermal desorption into a cryogenically cooled wide bore (0.53 mm i. d.) capillary column and oven temperature programming. The method has been routinely applied for profiling the mid-to-high boiling compounds (bp 80–225°C) in the headspace of a variety of foods and beverages. Method criteria, advantages and limitations are discussed. FID and NPD chromatograms for brewed coffee and peanut butter volatiles are presented as typical examples.     

Performance of fixed and fluidized bed reactors with immobilized enzyme

Applied Biochemistry and Biotechnology - Saccharification of α-amylase liquefied cassava starch was carried out at pH 4.5 and 45?C, in both fixed and fluidized bed reactors, using...     

Reticulated vitreous carbon electrode materials chemically modified with immobilized enzyme

The direct covalent attachment of glucose oxidase to the surface of reticulated vitreous carbon (RVC), a honeycomb carbonaceous material, is reported. The activity of the bound enzyme on different types of RVC, and the activity of adsorbed blanks are presented.     

Capillary electrophoresis mass spectrometry coupling with immobilized enzyme electrospray capillaries

Open tubular capillary enzyme reactors were studied for rapid protein digestion and possible on-line integration into a CE/ESI/MS system. The need to minimize the time of the analyte molecules to diffuse towards the surface immobilized enzyme and to maximize the surface-to-volume (S/V) ratio of the open tubular reactors dictated the use of very narrow bore capillaries. Extremely small protein amounts (atto-femtomoles loaded) could be digested with enzymes immobilized directly on the inside wall of a 10 microm I.D. capillary. Covalently immobilized L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-trypsin and pepsin A were tested for the surface immobilization. The enzymatic activity was characterized in the flow-through mode with on-line coupling to electrospray ionization-time of flight-mass spectrometer (ESI/TOF-MS) under a range of protein concentrations, buffer pH's, temperatures and reaction times. The optimized reactors were tested as the nanospray needles for fast identification of proteins using CE-ESI/TOF-MS.     

A strategy for screening trypsin inhibitors from traditional Chinese medicine based on a monolithic capillary immobilized enzyme reactor coupled with offline liquid chromatography and mass spectrometry

A novel strategy was successfully developed for screening trypsin inhibitors in traditional Chinese medicines based on monolithic capillary immobilized enzyme reactors combined with liquid chromatography‐tandem mass spectrometry. Organic polymer based monolithic enzyme reactors were firstly prepared by covalently bonding trypsin to a poly(glycidyl methacrylate‐co‐poly (ethylene glycol) diacrylate) monolith by the ring‐opening reaction of epoxy groups. The activity and kinetic parameters of the obtained monolithic trypsin reactors were systematically evaluated using micro‐liquid chromatography. Fourier transform infrared spectroscopy and scanning electron microscopy were also used to characterize the monolithic trypsin reactors. The resulting functional and denatured monolithic trypsin reactors were applied as affinity solid‐phase extraction columns, and offline coupled with a liquid chromatography‐tandem mass spectrometry system to construct a binding affinity screening platform. Subsequently, the proposed platform was applied for screening trypsin binders in a Scutellaria baicalensis Georgi extract. Three compounds, namely scutellarin, baicalin, and wogonoside were identified, and their inhibitory activities were further confirmed via an in vitro enzymatic inhibition assay. Additionally, molecular docking was also performed to study the interactions between trypsin and these three compounds.     

Development of dimethyl sulfoxide biosensor using a mediator immobilized enzyme electrode

A mediator immobilized dimethyl sulfoxide (DMSO) sensor using DMSO reductase (DMSO-R) was constructed. Methyl viologen (MV) was used as a mediator and immobilized on a glassy carbon (GC) electrode with Nafion polymer. DMSO-R from Rhodobacter sphaeroides f. sp. denitrificans was retained by a dialysis film on the modified GC electrode. The amperometric signal in response to DMSO was observed. The linear range of the calibration curve for DMSO was between 0 and 600 microM. The response time was within 100 s and the relative standard deviation was 4% at 200 microM DMSO (n = 4). To eliminate the background noise derived from oxygen in samples, the glucose oxidase-catalase retained DMSO sensor was also examined.     

Synthesis with immobilized enzyme of the most important sialic acid.

Starting from a crude mixture of N-acetylmannosamine and N-acetylglucosamine, N-acetylneuraminic acid has been easily synthesized on 2.8 millimoles scale, with one immobilized enzyme.     

Catalytic biofunctional membranes containing site-specifically immobilized enzyme arrays: a review

Biofunctional membranes normally involve the random immobilization of biomolecules to porous, polymeric membranes, often through the numerous lysine residues on the protein. In this process, bioactivity is significantly decreased largely due to different orientations of the biomolecule with respect to the membrane or to multiple point attachment. To circumvent this difficulty, while still taking advantage of the immobilization of biomolecules, site-specific immobilization of the biomolecule with the active (or binding) site directed away from the membrane is essential. In this review, we summarize our efforts involving biophysical and bioanalytical chemistry and chemical engineering, together with molecular biology, to develop and characterize such site-specifically membrane immobilized catalytic enzyme bioreactors. Site-directed mutagenesis, gene fusion technology, and post-translational modification methods are employed to effectuate the site-specific membrane immobilization. Electron paramagnetic resonance, in conjunction with active-site specific spin labels, kinetic analyses, and membrane properties are used to characterize these systems. Biofunctional membranes incorporating site-specifically immobilized biomolecules provide greater efficiency of biocatalysis, bioseparations, and bioanalysis.