Developments and applications of biosensors in food analysis.

The food industry needs suitable analytical methods for process and quality control; that is, methods that are rapid, reliable, specific and cost-effective in their provision of information about physical and chemical characteristics of food. Apart from a few important analytes, such as sugars, alcohols, amino acids, flavours and sweeteners, food applications mainly focus on the determination of contaminants. However, very few biosensors play a prominent role in food processing or quality control. Considerable effort must be made to develop biosensors that are inexpensive, reliable, and robust enough to operate under realistic conditions.

Enzyme reactions in the presence of cyclodextrins: biosensors and enzyme assays.

Cyclodextrins, macrocyclic carbohydrates with apolar internal cavities, can form complexes with, and solubilize many normally water-insoluble compounds. Ferrocene and its derivatives, tetrathiafulvalene and tetramethylbenzidine, can function as redox mediators, but are insoluble in water; when they are complexed with cyclodextrins, they can be used in enzymatic assays and in the construction of mediated biosensors. In addition, the solubilization of polynuclear aromatic hydrocarbons (PAHs), including the potent carcinogen benzo[a]pyrene, by cyclodextrins has enabled the detection of these important environmental contaminants.

Improvement of the selectivity of an FIA amperometric biosensor system for glucose.

A flow injection analysis (FIA) biosensor system has been developed for the determination of glucose from urine, blood plasma and foodstuffs. Glucose oxidase was immobilized onto porous aminopropyl glass beads via glutaraldehyde activation to form an enzyme column. The hydrogen peroxide released from the conversion of glucose to gluconic acid was monitored by a platinum electrode vs. silver/silver chloride poised at +700 mV. As a novel aspect to the improvement of the selectivity of the biosensor system, an anion exchange column was placed upstream to remove uric acid, ascorbic acid or acetaminophen, three major electroactive interfering substances which usually occur in urine and blood plasma. Among several resins tested, the effective adsorption of uric and ascorbic acids could be accomplished using an acetate anion exchanger, and the selectivity coefficient was pH dependent. The binding of acetaminophen to the resin was much less efficient and, in all cases, the selectivity coefficient was independent of the operating temperature up to 37 degrees C. When applied to real samples, the data obtained by the biosensor system compared well with those of the standard hexokinase assay. The immobilized glucose oxidase could be reused for at least 2000 repeated analyses without loss of its original activity.

An FIA biosensor system for the determination of phosphate.

A flow injection analysis (FIA) biosensor system for the determination of phosphate was constructed using immobilized nucleoside phosphorylase and xanthine oxidase and an amperometric electrode (platinum vs silver/silver chloride, polarized at 0.7 V). When a phosphate-containing sample was injected into the detection cell, phosphate reacted with inosine in the carrier buffer to produce hypoxanthine and ribose-1-phosphate in the presence of nucleoside phosphorylase. Hypoxanthine was then oxidized by xanthine oxidase to uric acid and hydrogen peroxide, which were both detected by the amperometric electrode. The response of the FIA biosensor system was linear up to 100 microM phosphate, with a minimum detectable concentration of 1.25 microM phosphate. Each assay could be performed in 5-6 min and the system could be used for about 160 repeated analyses. This system was applicable for the determination of phosphate in various food products and plasma, and the results obtained agreed well with those of the enzymatic assay.

The potential role of biosensors in the food and drink industries.

Despite their apparent potential as analytical tools in the food and drink industries, only a few biosensors are used routinely. This article describes the development of biosensors for these sectors and discusses the technical and economic problems of applying this technology to the monitoring of food and drink products.

A chemiluminescence fiber-optic biosensor system for the determination of glutamine in mammalian cell cultures.

A chemiluminescence fiber-optic biosensor system has been developed for determining glutamine in hybridoma cell cultures producing monoclonal antibodies against viral surface antigens. Glutaminase and glutamate oxidase (GLO) were immobilized onto aminopropyl glass beads via glutaraldehyde activation separately and packed in a column. Two separate columns containing immobilized GLO and catalase were placed upstream to eliminate endogenous glutamate. In the presence of ferricyanide, luminol reacted with hydrogen peroxide released from the enzymatic reactions to produce a chemiluminescence (CL) light signal which was detected and quantitated with a fiber-optic system. In combination with flow injection analysis it was possible to process samples virtually identically, thus avoiding difficulties in reproducing the CL signal. There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 10(-6) to 10(-3) M. A complete analysis could be performed in 2 min including sampling and washing. Each immobilized enzyme column was stable for at least 300 repeated analyses without any loss of activity. When the biosensor system was used for the determination of glutamine in spent mammalian cell cultures, the values obtained compared well with those of high-performance liquid chromatography, thus validating the applicability of the CL fiber-optic system.

Monitoring the activity of glucose oxidase during the cultivation of Aspergillus niger using novel amperometric sensor with 1, 1'-dimethylferricinium as a mediator.

1, 1'-dimethylferricinium (DMF+), a deep blue, and stable mediator, was prepared from a water-soluble 1, 1'-dimethylferrocene(DMF):2-hydroxypropyl- beta-cyclodextrin complex via enzymatic oxidation using immobilised bilirubin oxidase. This mediator was superior to other soluble ferrocenes, notably carboxyferrocene, in terms of both solubility (110 mM vs 0.5 mM) and oxidation potential (150 mV vs 300 mV against Ag/AgCl). Although the cyclic voltammogram of DMF+ was electrochemically equivalent to DMF, the use of the former resulted in a significantly lower background current (< 10 nA vs 30 nA). Because of its higher solubility, concentrated stock solutions of DMF+ can be prepared and supplied to the electrode. This is of particular importance when the signal is severely limited by the rate at which the working electrode can oxidase DMF to DMF+. A linear response of current versus units of glucose oxidase (GOD) was obtained up to 0.5 unit/ml. The detection limit was estimated to be 0.03 unit/ml and the response time was 2.5 min or less. The amperometric system was used successfully to follow the GOD activity during the growth of Aspergillus niger a well-known GOD producer. The results obtained correlated well with a standard absorbance-based assay using dichlorophenol-indophenol (DCPIP). The KM of GOD for the glucose in the lysate was measured as 38 mM. A reduced response and higher KM (48 mM) of the cell homogenate, compared to the lysate, illustrated the requirement for the DMF+ and glucose to diffuse across the cell membrane to interact with GOD in whole cells.

Derivatization, stabilization and detection of biogenic amines by cyclodextrin-modified capillary electrophoresis-laser-induced fluorescence detection.

o-Phthalaldehyde (OPA) derivatives of eight biogenic amines were stabilized at 5 degrees C by forming inclusion complexes with methyl-beta-cyclodextrin (MBCD). The derivatives were separated and detected by cyclodextrin-modified capillary electrophoresis (CE) with UV or laser-induced fluorescence (LIF) detection. Using a borate buffer, pH 9.0 consisting of ethanol and a mixture of negatively charged sulfobutylether-beta-cyclodextrin and neutral MBCD, baseline separation of the eight OPA derivatives was achieved within 25 min with high separation efficiencies. The detection limits (S/N=3) obtained by UV and LIF detection were determined to be 10 microM and 0.250 microM, respectively. Glutamic acid was added after the initial derivatization step to neutralize residual OPA which otherwise caused a significant interference, particularly when analysis was performed around the detection limit of the OPA derivatives. Important biogenic amines in fish, wine and urine were then derivatized and determined by CE-LIF. In the case of sole and rainbow trout, the results obtained were validated by an enzymatic assay using putrescine oxidase.

Determination of urinary glucose by a flow injection analysis amperometric biosensor and ion-exchange chromatography.

A practical biosensor system has been developed for the determination of urinary glucose using a flow-injection analysis (FIA) amperometric detector and ion-exchange chromatography. Glucose oxidase was immobilized onto porous aminopropyl glass beads via glutaraldehyde activation to form an immobilized enzyme column. On the basis of its negative charge at pH 5.5, endogenous urate in urine samples was effectively retained by an upstream anion-exchange resin column. The biosensor system possessed a sensitivity of 160 +/- 2.4 RU microM-1 (RU or relative unit is defined as 2.86 microV at the detection output) for glucose with a minimum detection level of 10 microM. When applied for the determination of urinary glucose, the result obtained compared very well with that of the widely accepted hexokinase assay. The immobilized glucose oxidase could be reused for more than 1000 repeated analyses without losing its original activity. The reuse of the acetate anion-exchange column before replacement would be about 25-30 analyses. Acetaminophen and ascorbic acid were also effectively adsorbed by the acetate anion exchanger. The introduction of this type of anion exchanger thus greatly improved the selectivity of the FIA biosensor system and fostered its applicability for the determination of glucose in urine samples.

An improved enzymatic assay for glucose determination in blood serum using a 1,1'-dimethylferricinium dye.

1,1'-dimethylferricinium (DMFe+), a stable and pH-insensitive blue dye, was prepared via enzymatic oxidation of a 1,1'dimethyl-ferrocene (DMFe):2-hydroxypropyl-beta-cyclodextrin (HPCD) water-soluble inclusion complex, using bilirubin oxidase immobilized onto porous aminopropyl glass beads via glutaraldehyde activation. In the presence of glucose, DMFe+ was reduced to DMFe by reacting with the reduced glucose oxidase (FADH2), and the absorbance decrease was followed at 650 nm. In acetate pH 5.2 buffer, the response to glucose in blood serum was nonlinear, especially in the low concentration range, because of a competition for the reduced glucose oxidase between the DMFe+ dye and oxygen. At this pH, endogenous ceruloplasmin was also observed to oxidize residual DMFe (16%) in the dye preparation, causing an increase in absorbance at 650 nm. An assay protocol was then developed using maleate buffer, pH 6.5, to overcome these interferences as well as mutarotation of alpha-D-glucose. The results obtained for glucose in the blood serum samples agreed well with those of the reference hexokinase/glucose-6-phosphate dehydrogenase method.

Application of a novel 1,1'-dimethylferricinium dye for the determination of uric acid in urine.

Water-soluble 2-hydroxypropyl-beta-cyclodextrin (Hp-beta-CyD), a cyclic and nonreducing oligosaccharide was used to enclose a hydrophobic guest molecule 1,1'-dimethylferrocene (DMF) to form a water-soluble yellow complex. At high concentrations (300 mM), Hp-beta-CyD enclosed up to 100 mM DMF. The yellow complex was electrochemically oxidized (platinum vs Ag/AgCl poised at +450 mV) to form a blue dye, 1,1'-dimethylferricinium (DMF+). This is a one-electron transfer process and the ferricinium cation formed exhibited an absorption peak at 650 nm. The concentrated DMF+ was stable for at least 4 mo at 4 degrees C and insensitive to a wide pH variation (pH 2-11). Application of the novel DMF+ complex as a colorimetric dye for the determination of uric acid in urine was successfully demonstrated. The reaction between the dye and uric acid is almost instantaneous and decrease in absorbance caused by the reduction of 1,1'-dimethylferricinium to 1,1'-dimethylferrocene can be followed at 650 nm. The results obtained agreed well with those of the reference reversed-phase HPLC method.

An improved FIA biosensor for the determination of aspartame in dietary food products.

A flow injection analysis (FIA) biosensor system was developed for the determination of the artificial sweetener aspartame (L-aspartyl-L-phenylalanine methyl ester). The system consisted of an enzyme column of pronase immobilized on activated arylamine glass beads and a L-amino acid oxidase electrode connected in series. The dipeptide bond of aspartame was cleaved by immobilized pronase to release phenylalanine, which was in turn monitored by the enzyme electrode that used L-amino acid oxidase immobilized on a preactivated nylon membrane in combination with an amperometric electrode (platinum vs silver/silver chloride, 700 mV). The response of the FIA biosensor was linear up to 1 mM aspartame with a lower detection limit of 25 microM and had good reproducibility (rsd 0.3%). The FIA biosensor was stable for at least 30 h of continuous use at Tr. Each assay takes 4 min giving a sample throughput of 15 h-1. When applied to aspartame in dietary food products the results obtained agreed well with those reported by the product manufacturers.

Regulation of coenzyme utilization by bovine liver glutamate dehydrogenase: investigations using thionicotinamide analogues of NAD and NADP in a dual wavelength assay.

1. The coenzyme preference of bovine liver glutamate dehydrogenase (GDH) was probed using dual wavelength spectroscopy and pairing the thionicotinamide analogues, S-NAD or S-NADP (which have absorbance maxima at 400 nm), with the natural coenzymes, NADP or NAD. 2. S-NAD and S-NADP were found to be good alternate substrates for GDH: the apparent Km's for the thioderivatives were similar to those of the corresponding natural coenzymes, the apparent Km's for glutamate were unaltered by the substitution of the thioderivatives, and the effects of inhibitors and activators on S-NAD or S-NADP kinetics were qualitatively the same as those found for NAD or NADP, respectively. 3. Dual wavelength assays paired NAD and S-NADP or S-NAD and NADP to study the simultaneous reduction of the two coenzymes. Conditions of increasing glutamate concentrations produced differential effects on the rates of the NAD vs NADP reactions, the result, with either nucleotide pair, promoting the NADP linked reaction. 4. Activators and inhibitors of the GDH reaction also showed differential effects upon the NAD vs NADP linked reaction rates in the dual wavelength assay. ADP and leucine, which activate both the NAD and the NADP linked reactions in single coenzyme assays, preferentially activate the NADP or S-NADP linked reactions in the dual nucleotide assays. GTP produced greater inhibition of the NAD or S-NAD linked reactions than of the NADP or S-NADP reactions while ATP inhibited NAD or S-NAD reactions and activated NADP or S-NADP reactions. The net effect of all metabolite modulators was to promote the NADP linked reaction by decreasing the activity ratios, v(NAD)/v(S-NADP) or v(S-NAD)/v(NADP). 5. The results are consistent with the suggestion that NADP is the preferred coenzyme for the oxidative deamination of glutamate by GDH even though the enzyme is capable of utilizing either coenzyme in vitro.

Synthesis and characterization of a water-soluble affinity polymer for trypsin purification.

A specific ligand bound polymer has been synthesized for the purpose of purification and stabilization of trypsin, an easily autodigestible enzyme. The affinity polymer was formed by copolymerizing N-acryloyl-m-aminobenzamidine, a strong trypsin inhibitor, and acrylamide in the absence of oxygen. Kinetic studies on the trypsin inhibition revealed that there was a strong binding between this enzyme and the polymer and the mechanism was of a competitive manner with an inhibition constant of 0.6 x 10(-3)M. Such an affinity polymer was also very effective in preventing trypsin from auto-digestion at 4 degrees C.Based on this finding and the principle of cross flow filtration, a new process has been developed for purification of trypsin from a solution containing chymotrypsin. The experimental data indicated that trypsin was bound to the polymer (MW > 10(5)) and remained in the retentate while unbound chymotrypsin was collected in the filtrate. This purification process has a capability of recovering 98% pure trypsin at 90% yield.

A continuous affinity ultrafiltration process for trypsin purification.

A continuous process has been devised and tested for purification of a crude trypsin preparation from pig pancreas. The development was based on the principles of affinity chromatography and Ultrafiltration. Trypsin was selectively attracted by a water-soluble high molecular weight (>100,000) polymer, bearing a potent and specific trypsin inhibitor, m-aminobenzamidine. The trypsin-macroligand complex was then retained by using an appropriate Ultrafiltration membrane, while impurities could pass through. The bound trypsin was eluted by either arginine or benzamidine. The process also featured provision for recirculation of the eluant as well as the macroligand. It was demonstrated that this purification process could purify trypsin from the crude preparation with a yield of 77%, contaminated with only 3% of impurities. For the first time, a serious attempt has been made toward continuous purification of enzymes by the affinity Ultrafiltration technique. Besides a substantial increase in productivity, the affinity polymer could be easily reconditioned and expected to possess a long operative life. Such characteristics undoubtedly will play an important role in reducing the cost of trypsin purification.

Electrochemical preparation of 1,1'-dimethylferricinium from a water-soluble 1,1'-dimethylferrocene-2-hydroxypropyl-beta-cyclodextrin complex and its applications in enzyme assay.

Water-soluble 2-hydroxypropyl-beta-cyclodextrin (hp-beta-CyD), a cyclic and nonreducing oligosaccharide, was used to enclose the hydrophobic guest molecules ferrocene (FeCp2) and 1,1'-dimethylferrocene (DMFeCp2) to form a water-soluble complex. At high concentrations (300 mM), hp-beta-CyD enclosed up to 100 mM FeCp2 or DMFeCp2. The yellow complexes were electrochemically oxidized (platinum vs Ag/AgCl poised at +450 mV) to form the blue dyes ferricinium (FeCp2+) and 1,1'-dimethylferricinium (DMFeCp2+). This is a one-electron transfer process and the ferricinium cations formed exhibited absorption peaks at 620 and 650 nm, respectively. The concentrated DMFeCp2+ was much more stable (4 months) than the FeCp2+ (2 days) and both oxidized dyes were insensitive to a wide pH variation (pH 2-11). The DMFeCp2+ was reduced easily by various reducing agents such as ascorbic acid, uric acid, and sulfite and exhibited an absorption coefficient of 325 cm-1 M-1. Application of the novel DMFeCp2+ complex as a colorimetric dye for the enzymatic oxidation of glucose, glutamate, lactate, phenylalanine, xanthine, and hypoxanthine was successfully demonstrated. When applied to real samples the results obtained agreed well with those of standard enzymatic assays. The DMFeCp2+ complex could also be utilized to monitor activity of oxidases under saturating substrate concentrations.

Isolation of urokinase by affinity ultrafiltration.

A water-soluble, ligand-bound polymer has been synthesized for the purpose of isolation of urokinase, an important plasminogen activator. The affinity polymer was formed by copolymerizing N-acryloyl-m-aminobenza-midicine and acrylamide in the absence of oxygen. An affinity ultrafiltration process was then developed for isolating urokinase from an artificial solution containing peroxidase and urokinase and from a crude urine source. The process yields were determined to be 86% and 49%, respectively. The recovered urokinase exhibited a specific activity close to that of the highest commercial grade. This article also presents a new technique for assaying urokinase by coupling plasminogen with L-benzoyl arginine-p-nitroanilide (L-BAPNA), an inexpensive chromogenic substrate.

Development of a biosensor for assaying postmortem nucleotide degradation in fish tissues.

An enzyme sensor system has been developed to assess the freshness level in fish tissue. The system was designed to measure the K value, the concentration ratio of [Hx + HxR] and [Hx + HxR + IMP], where Hx, HxR, and IMP are hypoxanthine, inosine and inosine-5'-monophosphate, respectively. The [Hx + HxR] concentration in tissue extract was measured by nucleoside phosphorylase and xanthine oxidase immobilized on a preactivated nylon membrane and attached to the tip of a polarographic electrode. The electrode amperometrically detected the products of degradation, hydrogen peroxide and uric acid. For determination of [IMP + HxR + Hx], IMP was first converted to HxR by nucleotidase immobilized on the wall of a polystyrene tube. The enzyme electrode consisting of nucleoside phosphorylase and xanthine oxidase provided excellent reproducible results for at least 40 repeated assays and immobilized nucleotidase was good for at least 40 assays as well. The K value for each sample could be determined in ca. 10 min. When applied to K value measurements in several fish meats, the results obtained agreed well with those obtained by the conventional enzymatic method.

A substrate recycling assay for phenolic compounds using tyrosinase and NADH.

A substrate recycling assay for phenolic compounds was developed using tyrosinase, a copper-containing enzyme, in excess NADH. The reaction of various phenols with the enzyme produced an o-quinone, which was then detected by recycling between reactions with the enzyme and NADH. The recycling of quinones by excess NADH to their original reduced forms prevented the problems of subsequent quinone polymerization and product inactivation which occur in nonrecycling assays. Absorbance measurements of the NADH consumption rate enhanced the assay sensitivity for catechol 100-fold compared to nonrecycling o-quinone detection, giving a detection limit of 240 nM. Fluorescence NADH monitoring permitted a 10-fold improvement over absorbance, with a detection limit of 23 nM. The recycling reaction was selective for o-quinones, and no interference was noted for p-quinones or quinoneimines. The two-step oxidation of phenols was observed as an initial lag phase (ca. 10 min), requiring a higher enzyme concentration to achieve the same sensitivity as that for catechol. The procedure was most useful for assaying catechol, 4-chlorocatechol, phenol, p-cresol, and 4-chlorophenol and may provide selective detection of these components in mixtures. Several other derivatives of catechols, including amine derivatives, were also detected, with relative sensitivity being related to substrate activity of the enzyme.

A Combined Chemical and Electrochemical Approach Using Bis(trifluoroacetoxy)iodobenzene and Glucose Oxidase for the Detection of Chlorinated Phenols.

A novel electrocatalytic approach using a chemical reaction and an enzymatic reaction has been developed for the measurement of 18 chlorophenol congeners, including highly chlorinated pollutants such as pentachlorophenol, 2,3,5,6-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, and several trichlorophenols. Chlorophenols were oxidized to chlorobenzoquinones with very high yields using bis(trifluoroacetoxy)iodobenzene in 0.1 M trichloroacetic acid, pH 1.5, at ambient temperature. UV-visible spectrophotometry, cyclic voltammetry, and HPLC have been used to characterize the reaction products and yields. Together with glucose oxidase immobilized on a working glassy carbon electrode (+0.45 V vs Ag/AgCl), chlorinated benzoquinones have been demonstrated to be efficient mediators in a glucose oxidase/glucose system. In this approach, glucose oxidase was readily reduced by excess glucose to provide a non-rate-limiting source of electron flow toward the electrode. The oxidation products of chlorophenols then recycled the reduced glucose oxidase to its active oxidative state, i.e., mediating the rate-limiting electron transfer from the enzyme to the electrode. At pH 3.5, linear behavior of the current response was observed up to 200 nM for all chlorophenol oxidation products. The detection limit of this method for both pentachlorophenol and 2,3,5,6-tetrachlorophenol was about 4 nM, which is close to the maximum allowable contamination level of pentachlorophenol in water samples (2.7 nM). The detection limit obtained for pentachlorophenol could also be considered superior to the result obtained with the PCP immunoassay technology (13.3 nM).