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PubMed2001

Genotyping short tandem repeats using flow injection and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Characterizing polymerase chain reaction (PCR) amplicons has been accomplished for the first time using flow injection analysis coupled to electrospray ionization mass spectrometry (ESI-MS). The PCR amplicons were amplified at the human tyrosine hydroxylase short tandem repeat locus from an individual homozygotic for the 9.3 allele. One product was amplified using Pfu polymerase and yielded a blunt-ended amplicon of 82 base-pairs (bp) in length. The second PCR product was amplified using Taq polymerase that resulted in an amplicon with cohesive termini of 82 bp plus either mono- or diadenylation. The two PCR amplicons were alternatively injected using a 0.5-microL loop at 2 microM for the Pfu amplicon and 1 microM for the Taq amplicon with a flow rate of 200 nL/min during data acquisition. Both PCR amplicons were accurately identified using mass measurements illustrating the compatibility of ESI-MS for genotyping short tandem repeat sequences and the potential for high-throughput genotyping of large PCR amplicons.

Alleles↗
PubMed2002

Plant tissue-based chemiluminescence flow biosensor for determination of unbound dopamine in rabbit blood with on-line microdialysis sampling.

A novel plant tissue-based chemiluminescence (CL) biosensor for dopamine combined with flow injection analysis is presented in this paper. The potato roots act as molecular recognition elements. Dopamine is oxidized by oxygen under the catalysis of polyphenol oxidase in the tissue column to produce hydrogen peroxide, which can react with luminol in the presence of peroxidase of potato tissue to generate CL signal. The CL emission intensity was linear with dopamine concentration in the range of 1x10(-5)-1x10(-7) g/ml and the detection limit was 5.3x10(-8) g/ml (3sigma) with a relative standard deviation of 1.7%. Combined with microdialysis sampling, the biosensor was applied to monitor the variation of dopamine level in the blood of rabbit after the administration of dopamine to demonstrate the favorable resolution and reliability of the system for in vivo on-line monitoring.

Animals↗
PubMedDate not supplied

Kalman filter based glucose control at small set points during fed-batch cultivation of Saccharomyces cerevisiae.

A glucose control system is presented, which is able to control cultivations of Saccharomyces cerevisiae even at low glucose concentrations. Glucose concentrations are determined using a special flow injection analysis (FIA) system, which does not require a sampling module. An extended Kalman filter is employed for smoothing the glucose measurements as well as for the prediction of glucose and biomass concentration, the maximum specific growth rate, and the volume of the culture broth. The predicted values are utilized for feedforward/feedback control of the glucose concentration at set points of 0.08 and 0.05 g/L. The controller established well-defined conditions over several hours up to biomass concentrations of 13.5 and 20.7 g/L, respectively. The specific glucose uptake rates at both set points were 1.04 and 0.68 g/g/h, respectively. It is demonstrated that during fed-batch cultivation an overall pure oxidative metabolism of glucose is maintained at the lower set point and a specific ethanol production rate of 0.18 g/g/h at the higher set point.

Algorithms↗
PubMed1999

On-line monitoring of glucose and/or lactate in a fermentation process using an expanded micro-bed flow injection analyser.

A novel flow injection biosensor system for monitoring fermentation processes has been developed using an expanded micro bed as the enzyme reactor. An expanded bed reactor is capable of handling a mobile phase containing suspended matter like cells and cell debris. Thus, while the analyte is free to interact with the adsorbent, the suspended particulate matter passes through unhindered. With the use of a scaled down expanded bed in the flow injection analysis (FIA) system, it was possible to analyse samples directly from a fermentor without the pretreatment otherwise required to extract the analyte or remove the suspended cells. This technique, therefore, provides a means to determine the true concentrations of the metabolites in a fermentor, with more ease than possible with other techniques. Glucose oxidase immobilised on STREAMLINE was used to measure glucose concentration in a suspension of dead yeast cells. There was no interference from the cell particles even at high cell densities such as 15 gm dry weight per litre. The assay time was about 6 min. Accuracy and reproducibility of the system was found to be good. In another scheme, lactate oxidase was covalently coupled to STREAMLINE for expanded bed operation. With the on-line expanded micro bed FIA it was possible to follow the fermentation with Lactobacillus casei.

Bioreactors↗
PubMed2002

Toward a fully integrated positive-pressure driven microfabricated liquid analyzer.

A versatile integrated analyzer with a flow-programmed injection strategy and multiwavelength detection is described with applications toward sampling, flow injection analysis, and capillary separations. Continuous near-real-time sampling is a major benefit of the flow-programmed injection technique. Injection volumes ranging from 250 pL to several microliters were made without electrophoretic flow. Multiwavelength grating light reflection spectroscopy (GLRS) and transmission absorbance spectroscopy were performed simultaneously in a detection volume of 150 pL. The utility of these detection methods for refractive index (RI) and absorbance detection in capillary channels is demonstrated through analysis of salt, indicator, and dyes. GLRS is a unique, selective, and path-length-independent technique for probing RI, absorbance, and other optical properties. A limit of detection (LOD) of 170 microM was achieved for GLRS interferometric detection of FD&C Red #3, which corresponded to 2.6 fmol of analyte in the 150-pL detection volume. A LOD of 2 mM for phosphate buffer, or 3 fmol in the 150-pL detection volume will also be demonstrated. A siloxane coating on the GLRS grating was employed as a sensing layer to probe interactions between the sample and stationary phase. The combined GLRS interferometric response provided insight into both optical and chromatographic properties of samples. Open tubular capillary liquid chromatography with multidimensional multiwavelength detection is demonstrated for the analysis of three food dyes. Separation efficiency, N, of 16,000 was achieved for an unretained dye peak eluting at 12 min. Integration of novel sampling and detection schemes makes this a broadly applicable liquid analyzer.

Journal Article↗
PubMed1999

Monitoring of low concentrations of glucose in fermentation broth.

A highly sensitive glucose sensor, operating in flow-injection analysis (FIA) mode, was developed for the detection of glucose in fermentation broth. The assay system is based upon the post-column reaction of the peroxide formed in the glucose-oxidase-catalysed reaction and subsequent spectrophotometric detection of the coloured product formed. The sensor system was characterised and calibrated using standard solutions, and later used for quantification of glucose in fermentation media. Two types of enzyme column were used: one operated in packed-bed mode and the other in expanded-bed mode. Both columns were integrated into a FIA system and were found to give good analytical results. Glucose concentrations as low as 0.1 mg/l and 5 mg/l could be detected in packed- and expanded-bed modes respectively. Glucose concentrations were measured during typical fed-batch fermentation conditions in this system, and the results are presented.

Biosensing Techniques↗
PubMed2002

Flow injection spectrophotometric determination of L-ascorbic acid in biological matters.

A method for spectrophotometric determination of L-ascorbic acid (vitamin C) in tissues by flow injection analysis is reported. The procedure is based on the oxidation of analyse with iron(III) and 2,2'-dipyridyl. Concentrations of vitamin C in the range of 0.5-20 ppm have been determined with a relative standard deviation 1.2% (n=15). The injection rate was 40 samples per h. The method was applied to the determination of L-ascorbic acid in rat's tissues (blood serum, brain, and liver) and compared favourably with an independent reference method based on spectrophotometry.

Animals↗
PubMed1994

Measurement of free and total hydroxyproline by automated flow injection of serum or urine samples from maintenance hemodialysis patients with renal osteodystrophy.

An automated measurement of total and free hydroxyproline in serum or urine is presented that uses flow injection analysis. After exclusion of nonspecific substances, hydroxyproline was oxidized by chloramine-T and L-cysteine with Ehrlich's reagent. The linearity obtained was from 3.8 mumole/L to 1.22 mmole/L with good precision (CV < 3%). Comparison of the proposed method with HPLC yielded r = 0.939 as the correlation coefficient. Reference intervals of free and total hydroxyproline are 1.4-9.7 mumole/L, 3.8-27.2 mumole/L for serum, and 10.0-72.5 mumole/L, 25.2-303.6 mumole/L for urine, respectively. Serum free and total hydroxyproline levels in renal osteodystrophy patients on maintenance hemodialysis (N = 71) were significantly higher than in controls (P < 0.0001). This method is superior to the use of HPLC with regard to stability of the color reaction. The measurement of serum free and total hydroxyproline is a useful marker for therapeutic observation of renal osteodystrophy patients.

Chronic Kidney Disease-Mineral and Bone Disorder↗
PubMed2005

A novel thermal biosensor based on enzyme reaction for pesticides measurement.

A novel thermal biosensor based on enzyme reaction for pesticides detection has been developed. This biosensor is a flow injection analysis system and consists of two channels with enzyme reaction column and identical reference column, which is set for eliminating the unspecific heat. The enzyme reaction takes place in the enzyme reaction column at a constant temperature (40 degrees C) realized by a thermoelectric thermostat. Thermosensor based on the thermoelectric module containing 127 serial BiTe-thermocouples is used to monitor the temperature difference between two effluents from enzyme reaction column and reference column. The ability of this biosensor to detect pesticides is demonstrated by the decreased degree of the hydrolytic heat in two types of thermosensor mode. The hydrolytic reaction is inhibited by 36% at 1 mg/L DDVP and 50% at 10 mg/L DDVP when cell-typed thermosensor is used. The percent inhibition is 30% at 1 mg/L DDVP and 42% at 10 mg/L DDVP in tube-typed thermosensor mode. The detection for real sample shows that this biosensor can be used for detection of organophosphate pesticides residue.

Animals↗
PubMed2006

Universal response in liquid chromatography using charged aerosol detection.

A new, empirical approach is introduced to correct for the varying response of aerosol-based detectors with the varying composition of the mobile phase during gradient elution in HPLC. A Corona charged aerosol detector was used in the experiments. The detector is characterized by a nearly universal response at a given, constant mobile-phase composition for sufficiently nonvolatile analytes. A second pump was used to deliver an exactly inverse gradient compared to the analytical HPLC system, and both flows were mixed in a tee piece before introduction to the Corona detector. The approach proposed made it possible to extend the universal response from isocratic to gradient elution conditions in HPLC, vastly improving the usefulness of this detection technique. The constant response of the detector obtained in this way was first demonstrated in flow injection analysis. Very similar calibration curves were obtained for six sulfonamide drugs after mobile-phase compensation. The approach was also applied to gradient elution with excellent results. The data were characterized by good precision ranging from 4% RSD at 10 mg/L to 1.6% RSD at 780 mg/L. The average limit of detection with a 2-microL injection was 0.5 mg/L, corresponding to 1 ng injected on the column. The approach proposed allows quantification of unknown compounds, e.g., in pharmaceutical mixtures. Measurement of analytes at a relative concentration of 0.05% versus the main component is demonstrated.

Aerosols↗
PubMed1993

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.

Aspartame↗
PubMed2001

An integrated process for measuring the physicochemical properties of drug candidates in a preclinical discovery environment.

Automated log P, pK(a), solubility, and chemical stability systems comprise an integrated process that provides early stage physicochemical property data to the discovery research organization. Capillary electrophoresis (CE) techniques are used to experimentally determine pK(a) and log P. Solubility is determined using a quasi-equilibrium approach employing sample quantitation by flow injection analysis with ultraviolet (UV) detection at 256 nm. Chemical stability is assessed by challenging compounds with pH 2, pH 7, pH 12, and 3% hydrogen peroxide solutions overnight, and comparing the chromatographic profiles of the stability challenged solutions to that of a freshly prepared control. Validation of the log P method using a set of drug-like compounds demonstrates that the method yields log P values within +/-0.5 units of literature values. The log P method is valid over the range -0.5-5.0, and the technique is compatible with acidic, neutral, and basic compounds. The pK(a) technique yields results within +/-0.2 units of corresponding values obtained by potentiometric titration over a pK(a) range of 2 to 12. Solubility is reported in a 3-60 microg/mL range, and the results are generally within 20% of values measured by equilibrium solubility techniques. The current level of automation supports the measurement of the physicochemical properties of 100 compounds per week. Physicochemical property data for approximately 2000 compounds have been generated to date.

Automation↗
PubMed2001

Comparison of different protein immobilization methods on quartz crystal microbalance surface in flow injection immunoassay.

In this study, a quartz crystal microbalance (QCM) system operated repetitively in flow injection analysis (FIA) mode, is reported. Four immobilization approaches of seven different methods include: (i) physical adsorption; (ii) two thioamine thiolation methods, using cysteamine and cystamine for gold chemisorption and further coupling; (iii) two oxidized dextran spacer methods, coupling of cysteamine and cystamine thiolated QCM surface with periodate-oxidized dextran for further Schiff acid-base reaction; and (iv) two thiol-gold chemisorption-based self-assembled monolayer (SAM), applying short-chain, C(3), and long-chain, C(11), mercapto fatty acids to insolubilize human serum albumin (HSA) on QCM surface. Effects of these protein immobilization methods on FIA immunoassay of anti-HSA were compared. At the 0.01 mg/ml anti-HSA level, the lowest analyte concentration tested, the SAM using 11-mercaptoundecanoic acid as QCM surface activating agent generated a larger frequency shift than the other immobilization methods. This implied that the use of thiolated long-chain fatty acid constructed as self-assembled monolayer may thereby potentially be a useful protein immobilization method in QCM-FIA application.

Absorption↗
PubMed1993

A combined cellobiose oxidase/glucose oxidase biosensor for HPLC determination on-line of glucose and soluble cellodextrines.

Highly specific biosensors can be prepared by immobilizing flavin-containing oxidases in a redox polymer on an electrode surface. By combining one glucose oxidase and one cellobiose oxidase electrode in a flow cell we have made a sensor for flow-injection analysis, or postcolumn quantification, of glucose, cellobiose, and higher cellodextrines in an HPLC system. Samples of different concentrations of glucose and cellobiose, separately or mixed, were injected into the mobile phase and the current response was recorded simultaneously from both electrodes. The recorded response peak heights could be used for calibration curves. The usable measuring ranges were roughly 50 microM-50 mM for glucose and 5 microM-80 mM for cellobiose. Soluble cellodextrines, Glc1-6, could be separated on a C18 column by isocratic elution and detected by the sensor.

Biosensing Techniques↗
PubMed2006

Determination of riboflavin by enhancing the chemiluminescence intensity of peroxomonosulfate-cobalt(II) system.

A weak chemiluminescent (CL) emission was observed in the decomposition of peroxomonosulfate (HSO5-), which would be accelerated in the presence of trace amounts of cobalt (II). The mechanism was due to the production of singlet oxygen (1O2). Interestedly, riboflavin can enhance the CL and the CL intensity was strongly dependent on riboflavin concentration. Based on this phenomenon, a flow injection analysis (FIA) CL method was established for the determination of riboflavin. Additionally, the possible CL mechanism is proposed based on the kinetic curve of the CL reaction, CL spectra, UV-vis spectra and fluorescent spectra. The CL intensity was correlated linearly with concentration of riboflavin over the range of 1.0x10(-4) to 1.0x10(-8) g mL-1; the detection limit was 9.0x10(-9) g mL-1(S/N=3); the relative standard deviation was 1.4% for 9x10(-7) g mL-1 riboflavin (n=11). Furthermore, this method was applied to the determination of riboflavin in real tablets and injections successfully.

Cobalt↗
PubMed1997

Evaluation of a miniaturized thermal biosensor for the determination of glucose in whole blood.

A miniaturized thermal biosensor has been evaluated as part of a flow-injection analysis system for the determination of glucose in whole blood. Glucose was determined by measuring the heat evolved when samples containing glucose passed through a small column with immobilized glucose oxidase and catalase. Samples of whole blood (1 microliter) can be measured directly, without any pretreatment. The correlation in the response between the thermal biosensor, the Reflolux S meter (Boehringer Mannheim), the Granutest 100 glucose test kit (Merck Diagnostica) and the Ektachem (Kodak) instrument was evaluated. The influence of the hematocrit value and of possible interferences is reported. The correlation measurements show that the thermal biosensor calibrated with aqueous glucose standards generally gives lower values on blood glucose than the reference methods calibrated for serum or blood measurements. Mean negative biases range from 0.53 to 1.16 mmol/l. Differences in sample treatment clearly complicate comparisons and the proper choice of reference method. There was no influence from substances such as ascorbic acid (0.11 mmol/l), uric acid (0.48 mmol/l), urea (4.3 mmol/l) and acetaminophen (0.17 mmol/l) on the response to 5 mmol/l glucose. The hematocrit value does not influence the glucose determination, for hematocrit values of between 13 and 53%.

Biosensing Techniques↗
PubMed2002

Detection of glucose, galactose, and lactose in milk with a microdialysis-coupled flow injection amperometric sensor.

A microdialysis-coupled flow injection amperometric Sensor (microFIAS) was used to determine glucose, galactose, and lactose in milk. The sensor is based on enzyme-catalyzed reaction in combination with the three well-established analytical techniques, namely; microdialysis sampling, flow injection analysis (FIA), and amperometric detection. With the multianalyte sensor it was possible to detect glucose and galactose by sequential injection of their corresponding oxidase enzymes: glucose oxidase and galactose oxidase, while lactose was determined by injection of a mixture of beta-galactosidase and glucose oxidase enzymes. The sensor showed a linear response between 0.05 and 10 mM for glucose, between 0.1 and 20 mM for galactose and between 0.2 and 20 mM for lactose, respectively. The relative standard deviation values of the sensor measurements for glucose, galactose, and lactose were 3-4% (n = 3). The sensor measurements for lactose content in milk were compared with a standard method with an infrared spectrophotometer.

Animals↗
PubMed1993

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.

Acetaminophen↗