Surface plasmon resonance-based immunoassay for human C-reactive protein.

A rapid and highly-sensitive surface plasmon resonance (SPR)-based immunoassay (IA) has been developed and validated for detecting human C-reactive protein (CRP), a specific biomarker for inflammatory and metabolic disorders, and infections. The 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-activated protein A/G (Pr A/G) was diluted in 1% (v/v) 3-aminopropyltriethoxysilane (APTES), dispensed on a KOH-treated gold (Au)-coated SPR chip, and incubated for 30 min. The Pr A/G functionalized Au SPR chip was then bound to anti-human CRP capture antibody (Ab), blocked with bovine serum albumin, and subsequently used for the detection of CRP. The highly-simplified oriented Ab immobilization strategy enabled the leach-proof binding of capture Ab in 5-fold shorter time than conventional procedures. The developed IA detected 1.2-80 ng mL(-1) of CRP with a limit of detection (LOD) and a limit of quantification (LOQ) of 1.2 ng mL(-1) and 4.6 ng mL(-1), respectively. It detected CRP spiked in diluted human whole blood, serum and plasma as well as the CRP levels in the ethylenediaminetetraacetic acid (EDTA) plasma samples of patients with the same precision as the clinically-accredited analyzer-based IA and conventional CRP sandwich ELISA. The Ab-bound SPR chips stored at 4 °C retained their functional activity for 10 weeks, resulting in significant reduction in the overall analysis time.

Surface plasmon resonance-based immunoassay for human fetuin A.

This article describes a highly-sensitive surface plasmon resonance (SPR)-based immunoassay (IA) for human fetuin A (HFA), a specific biomarker for atherosclerosis and hepatocellular carcinoma. The assay is based on a novel immobilization procedure that simply involves the dilution of an anti-HFA capture antibody (Ab) in 1% (v/v) 3-aminopropyltriethoxysilane (APTES), followed by its dispensing on a KOH-treated gold (Au)-coated SPR chip and incubation for 30 min. The developed SPR IA detected 0.3-20 ng mL(-1) of HFA with a limit of detection and sensitivity of 0.7 ng mL(-1) and 1 ng mL(-1), respectively. The highly-simplified Ab immobilization procedure is also 5-fold more rapid than conventional procedures. It leads to the leach-proof binding of the capture Ab, which means that the developed SPR IA is highly cost-effective, as the Ab-bound SPR chip could be reused for many repeated HFA IAs after regeneration with 10 mM glycine-HCl, pH 2.0. The Ab-bound SPR chip, stored at 4 °C, lost only 18% of its original activity after 4 months. For the detection of HFA spiked in diluted human whole blood and plasma, the results obtained by the developed SPR IA agreed well with the commercial HFA sandwich ELISA.

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.

A microbial biosensor for trimethylamine using Pseudomonas aminovorans cells.

A biosensor system based on the difference in the oxygen uptake response of two microbial electrodes was developed to monitor trimethylamine (TMA). The first electrode, constructed using Pseudomonas aminovorans grown on TMA, was sensitive to TMA, trimethylamine N-oxide (TMAO), dimethylamine (DMA) and monomethylamine (MMA). The second electrode responding to TMAO, DMA and MMA was prepared using Ps. aminovorans grown on TMAO. The difference in oxygen uptake was linearly related to the TMA concentration in the range of 5-26 microM. The minimum detectable level was 2.6 microM and the relative standard deviation was determined to be 14% for 16 repeated analyses. When operated and stored at 30 degrees C, the response of the system was stable for only 2 days. However, when the biosensor system was operated at 30 degrees C but stored overnight at 4 degrees C, the system was stable up to 20 days. The biosensor system was applicable for the determination of TMA in fish tissue extracts and the results compared well with those determined by HPLC.

Monitoring glutamine in mammalian cell cultures using an amperometric biosensor.

An amperometric biosensor has been developed for monitoring glutamine in the pulsed-batch cultivation of murine hybridoma cells. Glutamine oxidase was cross-linked with bovine serum albumin (BSA) via glutaraldehyde activation and deposited on a preactivated nylon membrane. Glutaminase was then immobilized on the protein layer and the resulting membrane was attached to the sensing area of a hydrogen peroxide probe (platinum vs silver/silver chloride polarized at +0.7 V). An orthogonal test was performed to optimize the activity of the membrane for glutamine with respect to the concentrations of glutamate oxidase, BSA, glutaminase and glutaraldehyde. There was an excellent linear relationship between the biosensor's response and glutamine in the range 0.1-3 mM. The determination of glutamine could be performed in 2 min and each membrane was reused for at least 300 consecutive analyses. The data obtained also agreed well with those high-performance liquid chromatography, thus validating the applicability of the biosensor.

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.

Achiral selectivity in cyclodextrin-modified capillary electrophoresis.

Torus-shaped, circular, and hydrophilic cyclodextrins (CD) have been frequently used in capillary electrophoresis (CE) as buffer modifiers to effect chiral separation of enantiomers of drugs and specialty chemicals. Although less common, both neutral and charged cyclodextrins have also been exploited in CE to optimize the achiral separations of peptides, proteins, small molecules and a variety of positional isomers. Nonionic CDs are only useful for separations of net charged analytes through judicious partitioning of such guest molecules into their hydrophobic cavity of the former. However, they can be used with a surfactant for an effective resolution of neutral solutes as a result of a differential partitioning of such solutes in the micellar and the cyclodextrin-modified buffer phase. Ionic cyclodextrins, particularly, negatively charged derivatives with their own electrophoretic mobilities, increase the separation window and enable better resolution of analytes which weakly complex with or are poorly differentiated by neutral cyclodextrins.

Determination of explosives in soil and ground water by liquid chromatography-amperometric detection.

Electrochemical reduction of trinitrotoluene (TNT) and several nitroaromatics has been exploited toward the development of an amperometric detector for liquid chromatography (LC). Up to a ten-fold increase in sensitivity was accomplished for the explosives using amperometric detection instead of conventional UV measurement. A working glassy carbon electrode (poised at -0.80 V vs. Ag/AgCl) offered a detection limit of 9, 44 and 550 nM for trinitrobenzene, TNT and 1,4-dinitrobenzene, respectively. Separation of eleven TNT-related compounds in a mixture was achieved within 15 min using a C18 column and a mobile phase consisting of acetonitrile-50 mM phosphate buffer pH 5 (1:2, v/v) and 18 mM sodium dodecylsulfate. The LC-amperometric detection system was applicable for analyzing soil extracts and ground water and the results obtained agreed well with that of the US Environmental Protection Agency recommended procedure. Extension to analysis of HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) was accomplished with a silver working electrode instead of a glassy carbon electrode installed in a thin channel cell.

Nonaqueous capillary electrophoresis equipped with amperometric detection for analysis of chlorinated phenolic compounds.

Nonaqueous capillary electrophoresis (NACE) equipped with amperometric detection has been developed for separation and detection of an 11-member model mixture of chlorinated phenolic compounds. With triacetyl-beta-cyclodextrin (TACD) as a novel selectivity selector, acetonitrile proved to be an excellent solvent for this water-insoluble cyclodextrin derivative. Resolution of the analytes was achieved by using an optimized acetonitrile medium consisting of 500 mM acetic acid, 10 mM sodium acetate, 12 mM TACD and 50 mM tetrabutylammonium perchlorate. Separation of analytes was attributed to differential electrostatic and/or inductive interactions of the analytes with the TACD/TBA+ complex and charged tetrabutylammonium phases. A simple end-column amperometric detector (Pt vs. Ag/AgCl, poised at +1.6 V) in conjunction with NACE was used to analyze chlorophenols. Amperometric detection of such target compounds in acetonitrile-based media offers high sensitivity and alleviates electrode fouling compared to aqueous buffers. The detection limits obtained, ranging from 30 nM to 500 nM, are 3-8-fold lower than those obtained with aqueous buffers.

Adaptive control at low glucose concentration of HEK-293 cell serum-free cultures.

Fed-batch cultures were implemented to study the metabolism of HEK-293 cells. Glucose, measured every 30 min by a FIA biosensor system, was maintained at 1 mM throughout the culture using an adaptive nonlinear controller based on minimal process modeling. The controller performed satisfactorily at both low and high cell concentrations without the need for retuning between different culture phases. Overall, lactate production was significantly reduced by maintaining a low glucose concentration, thus decreasing the rate of glycolysis. The rates of glucose and glutamine uptake as well as the lactate and ammonia production were compared to those obtained in batch mode with an initial glucose concentration of 21 mM. Basically, three phases were observed in both culture modes. The metabolic shift from the first to the second phase was characterized by a significant reduction in glucose consumption and lactate production while maximum growth rate was maintained. The specific respiration rate appeared unchanged during the first two phases, suggesting that no change occurred in the oxidative pathway capacity. In the third phase, cell growth became slower very likely due to glutamine limitation.

Utilization of two improved enzyme immunoassays based on avidin-biotin interaction for the detection of Salmonella.

Based on a strong interaction between avidin and biotin, two enzyme immunoassays have been modified and tested for the detection of Salmonella typhimurium in foodstuffs. In both assays, the antigen containing sample was first reacted with antibody to Salmonella which was precoated on a polystyrene microtiter plate. The bound antigen was then allowed to react with an appropriate amount of biotinylated antibody. In the first procedure, the presence of Salmonella was quantified by using peroxidase-labeled avidin. In the latter, avidin acted as a bridge between the biotinylated antibody and the biotinylated peroxidase. Samples containing 10(3) and 10(4) cells/ml of the Salmonella virulent strain, respectively, were detectable by these two methods. The results thus compared favorably with the detection limit of the standard ELISA (10(5) cells/ml). The superiority of the modified ELISA's utilizing biotin/avidin interactions was also demonstrated for the detection of Salmonella in artificially contaminated food samples inoculated with only 2-5 Salmonella cells followed by two incubation steps. No significant interference of E. coli (up to 5 x 10(6) cells/ml) was observed in the quantification of Salmonella cells.

Development of a flow injection analysis (FIA) immunosensor for the detection of Escherichia coli.

A flow injection immunoanalysis (FIA) system has been developed for the detection of Escherichia coli in artificially contaminated food samples. Anti-E. coli antibodies were covalently immobilized onto porous aminopropyl glass beads via glutaraldehyde activation to form an immunoreactor. After adsorption of the cells onto anti-E. coli antibody bound glass beads, 4-methylumbelliferyl-beta-D-glucuronide was injected into the system which was then hydrolyzed by the adsorbed E. coli cells containing beta-D-glucuronidase, an enzyme which is very specific to E. coli and to a few other strains of Shigella. Fluorescent 4-methylumbelliferone released from the enzymatic reaction was then detected by a fluorometer. Owing to the specificity of the antibody towards E. coli, the FIA system was very selective for detection of E. coli whereas Shigella boydii, another GUD-positive bacterium, did not give any response. The FIA system was successfully used for detecting as low as 5 x 10(7) CFU/ml E. coli in less than 30 min and was reusable for at least 300 repeated assays. The immunoreactor yielded reproducible results during 3 months of experimentation if stored overnight at 4 degrees C in carrier buffer containing 0.05 to 0.25% Tween 20.

On-line chemiluminescence assay using FIA and fiber optics for urinary and blood glucose.

A chemiluminescence fiber optic system coupled to flow injection analysis (FIA) and ion exchange chromatography has been developed for determining glucose in blood and urine. Immobilized glucose oxidase acted on beta-D-glucose to produce hydrogen peroxide, which was then reacted with luminol in the presence of ferricyanide to produce a light signal. Endogenous ascorbic acid and uric acid present in urine or blood samples were effectively retained by an upstream acetate anion exchanger. In addition, acetaminophen could also be adsorbed by this ion exchanger. The detection system exhibited a sensitivity of 1.315 +/- 0.044 RU microM-1 for glucose with a minimum detection level of 1 microM. When applied for the determination of urinary and blood glucose levels, the results obtained compared well with those of the reference hexokinase assay. Immobilized glucose oxidase was reused for over 500 analyses without losing its original activity. A conservative estimate for the reuse of the acetate ion exchange column was about 100 analyses.

The development and application of a new affinity partitioning system for enzyme isolation and purification.

A reactive water-soluble polymer was synthesized by copolymerizing N-isopropylacrylamide and glycidyl acrylate. The reactive polymer could react with the amino groups of enzymes/proteins or other ligands to form an affinity polymer. As a model, the reactive polymer was allowed to react with paraaminobenzamidine, a strong trypsin inhibitor. The affinity polymer could easily form an aqueous two-phase system with either dextran or pullulan, and the phase diagram was compared favorably to that of the well-known polyethylene glycol-dextran system. Once trypsin was attracted to the affinity polymer dominant phase, the enzyme could be dissociated from the polymer at low pH. Owing to the N-isopropylacrylamide units, the affinity polymer could be isolated from the solution by precipitation at a low level of ammonium sulfate. The enzyme recovery was always greater than 50%, and the affinity polymer could be reused in several cycles of affinity partitioning and recovery.