University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Bo Mattiasson

Abbrev:
Mattiasson, B.
Other Names:
Address:
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.B. 124, SE- 22100, Lund, Sweden
Phone:
+46 46 2228264
Fax:
+46 46 2224713

Citations 32

"An Automated Flow Injection Analysis System For On-line Monitoring Of Glucose And L-lactate During Lactic Acid Fermentation In A Recycle Bioreactor"
World J. Microbiol. Biotechnol. 2001 Volume 17, Issue 1 Pages 23-29
M.A. Kumar, M.S. Thakur, A. Senthuran, V. Senthuran, N.G. Karanth, R. Hatti-Kaul, B. Mattiasson

Abstract: The study concerns on-line sequential analysis of glucose and L-lactate during lactic acid fermentation using a flow injection analysis (FIA) system. Enzyme electrodes containing immobilized glucose oxidase and L-lactate oxidase were used with an amperometric detection system. A 12-bit data acquisition card with 16 analog input channels and 8 digital output channels was used. The software for data acquisition was developed using Visual C++, and was devised for sampling every hour for sequential analyzes of lactate and glucose. The detection range was found to be 2-100 g L-1 for glucose and 1-60 g L-1 for L-lactate using the biosensors. This FIA system was used for monitoring glucose utilization and L-lactate production by immobilized cells of Lactobacillus casei subsp. rhamnosus during a lactic acid fermentation process in a recycle batch reactor. After 13 h of fermentation, complete sugar utilization and maximal L-lactate production was observed. A good agreement was observed between analysis data obtained using the biosensors and data from standard analyzes of reducing sugar and L-lactate. The biosensors exhibited excellent stability during continuous operation for at least 45 days.
Glucose

"Evaluation Of Direct Versus Competitive Binding Assays In Flow Injection Systems"
Process Control Qual. 1992 Volume 4, Issue 1 Pages 37-45
M. Nilsson, H. Håkanson and B. Mattiasson

Abstract: A small column of immobilized concanavalin A was place in a FIA system and used to carry out direct and indirect binding analyzes using horseradish peroxidase. In the latter type of assay, addition of a free carbohydrate, methyl αD-mannopyranoside, provided the competitive binding situation. The direct binding assays gave straight calibration curves and good fits in gradient experiments. The competitive assay gave more complex calibration curves with more complex data evaluation and less precision at higher concentrations. When suitable it is more straightforward to utilize a direct binding assay for process control in the production of enzymes.
Immobilized enzyme Gradient technique Process control

"Online Monitoring Of Product Concentration By Flow-ELISA In An Integrated Fermentation And Purification Process"
J. Ferment. Bioeng. 1994 Volume 78, Issue 5 Pages 356-360
Mats Nilsson, Arumugaswamy Rajendran Vijayakumar, Olle Holst, Christina Schornack, Håkan Håkanson and Bo Mattiasson*

Abstract: Process integration involving fermentation with cell recycling, affinity adsorption to remove the product, and online process monitoring of the product were investigated. α-Amylase was produced in continuous and batch fermentations of Bacillus amyloliquefaciens with complete cell recycling, which was achieved using a membrane unit coupled to the fermenter. An affinity column of cross-linked starch was employed in the recycling loop to adsorb the α-amylase before the cell-free broth was recycled to the fermenter. The concentration of α-amylase in the cell-free broth prior to and after the affinity separation step was continuously registered using a flow injection, immunochemical monitoring system. The different unit operations were integrated pair-wise, and eventually as a complete system. The complete feasibility of monitoring a macromolecular product online during a cultivation/adsorption process was also demonstrated.
α-Amylase Fermentation broth Process monitoring

"Process Monitoring By Flow Injection Immunoassay. Evaluation Of A Sequential Competitive-binding Assay"
J. Chromatogr. A 1992 Volume 597, Issue 1-2 Pages 383-389
Mats Nilsson, HÅakan HÅakanson and Bo Mattiasson

Abstract: The system used was similar to that described previously (Anal. Chim. Acta, 1991, 249, 163). Three proteins were determined on a column of rabbit anti-human serum albumin (HSA) antibodies covalently linked to Sepharose C1-4B. The sample containing the antigen (e.g., HSA) was passed through the column followed by a solution of the antigen - enzyme conjugate (for HSA the enzyme was peroxidase). After the column had been washed with buffer the enzyme activity was measured with a pulse of substrate, e.g., 14 mM phenol - 0.8 mM 4-aminoantipyrine - 9.3 mM H2O2. The eluate was monitored at wavelengths depending on the antigen and substrate. Calibration graphs for HSA and IgG were non-rectilinear, but were usable to concentration. of 200 and 400 mg l-1, respectively. A new variation on the theme of flow injection binding assays is presented, namely the sequential competitive binding assay, in which the sample containing the native antigen is first introduced into the flow system followed by a pulse of labeled antigen. The flow injection binding assay was used in monitoring the effluent from column chromatography sepns. of proteins. By applying a computer-based evaluation system, concentrations. are automatically read and compensation is made for denaturation in the affinity sorbent.
Proteins Immunoassay Immobilized antibody Sepharose beads Process monitoring

"Amperometric Biosensor For The Detection Of Hydrogen Peroxide Using Catalase Modified Electrodes In Polyacrylamide"
J. Biotechnol. 2005 Volume 119, Issue 2 Pages 172-180
Shailly Varma and Bo Mattiasson

Abstract: A simple biosensor for the detection of hydrogen peroxide in organic solvents has been developed and coupled to a flow injection analysis (FIA) system. Catalase was entrapped in polyacrylamide gel and placed on the surface of platinum (working electrode) fixed in a Teflon holder with Ag-wire (auxiliary electrode), followed by addition of filter paper soaked in KCl. The entrapped catalase gel was held on the electrode using membranes. The effects of cellulose and polytetrafluoroethylene (PTFE) membranes on the electrode response towards hydrogen peroxide have been studied. The modified electrode has been used to study the detection of hydrogen peroxide in solvents like water, dimethyl sulfoxide (DMSO), and 1,4-dioxane using amperometric techniques like cyclic voltammetry (CV) and FIA. The CV of modified catalase electrode showed a broad oxidation peak at -150 mV and a clear reduction peak at -212 mV in the presence of hydrogen peroxide. Comparison of CV with hydrogen peroxide in various solvents has been carried out. The electrode showed an irreversible kinetics with DMSO as the solvent. A flow cell has been designed in order to carry on FIA studies to obtain calibration plots for hydrogen peroxide with the modified electrode. The calibration plots in several solvents such as water, dimethyl sulfoxide, 1,4-dioxane have been obtained. The throughput of the enzyme electrode was 10 injections per hour. Due to the presence of membrane the response time of the electrode is concentration dependent.

"Automated Immunochemical Binding Assay (flow-ELISA) Based On Repeated Use Of An Antibody Column Placed In A Flow Injection System"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 381-394
Mats Nilsson, Gustav Mattiasson and Bo Mattiasson**

Abstract: A computer-controlled immunological binding assay intended for monitoring concentrations of interesting metabolites during bioprocesses has been developed. The main focus has been on speed, reliability, reproducibility and operational stability of the assay. The assay is based on the repeated use of a preparation of immobilized antibodies. Stability in the regeneration step is a prerequisite and was achieved by intermittent recalibration of the system. Furthermore, before immobilizing the polyclonal antibodies, they were purified on immobilized antigen and eluted using the same procedure as used in the assay cycle. Antibody preparation could be used for more than 50 cycles and in some cases for several hundred cycles. Since the calibration curve is valid for the whole life-time of the antibody column, recalibration only involved the registration of the column capacity. Experiments have been performed concerning monitoring of chromatographic separations as well as adsorption experiments from complex mixtures.
Immunoassay Biotechnology Column

"A Simple Spectrophotometric Method Based On PH-indicators For Monitoring Partial And Total Alkalinity In Anaerobic Processes"
Environ. Technol. 2003 Volume 24, Issue 9 Pages 1061-1067
T. G. Jantsch & B. Mattiasson

Abstract: Partial alkalinity, as a measure of bicarbonate concentration, and total alkalinity, as a measure of the concentration of bicarbonate and volatile fatty acids, are useful parameters for monitoring anaerobic digestion processes. This paper reports a new method based on pH-indicators and spectrophotometric measurements for the monitoring of partial and total alkalinity. The method was used in an off-line procedure for monitoring of an anaerobic process and displayed less than 5% deviation from the traditional method of measuring partial and total alkalinity, as well as being rapid and reproducible. A flow injection analysis system based on the method was used on-line for monitoring overload in a UASB reactor, which demonstrated changes in alkalinity not easily registered using traditional methods.

"A Low Temperature Microbial Biosensor Using Immobilised Psychrophilic Bacteria"
Biotechnol. Techniq. 1999 Volume 13, Issue 10 Pages 689-693
Renu Nandakumar, Bo Mattiasson

Abstract: A psychrophilic bacteria, Deinococcus radiodurans, was used to construct a biosensor to be used in a flow injection system. The transducer used was an O-2 electrode. The response of this cell-based electrode was studied towards a number of sugars. The temperature dependence of the electrode response correlated well with the behavior of the cells. Thus, the optimum temperature for measurement of glucose (0.55 mM) was about +5°C. Since the organism used is psychrophilic, a response time at this low temperature is similar to what is achieved with mesophilic organisms at room temperature. This is the first biosensor constructed using a psychrophilic microorganism.

"Variations In Plasmid Content During Escherichia Coli Cultivations Detected By On-line Flow Injection Processing"
Biotechnol. Lett. 2001 Volume 23, Issue 14 Pages 1135-1140
M.P. Nandakumar, E. Nordberg Karlsson, B. Mattiasson

Abstract: An integrated flow injection process for analysis of intracellular components of microbes has been used to monitor plasmid content in Escherichia coli cultivations inoculated with cells subcultured in the presence or absence of ampicillin. The system allows sampling, sample handling, cell disruption, separation of intracellular components, and analysis in a semi-on-line mode of operation. The time scale for the assay is in the range 15 min (plasmid peak) to 25 min (complete assay cycle). As expected, lower initial plasmid content was found using an inoculum subcultured in the absence of ampicillin. More importantly, significant decrease in plasmid content was detected in the later stages of the cultivations (grown in ampicillin containing medium) even when using inoculum subcultured in the presence of ampicillin. This illustrates the versatility of the system, which allows monitoring of plasmid content as the cultivation proceeds.

"Fluorimetric Quantification Of Intracellular Lactate Dehydrogenase During Fermentation Using Flow Injection Analysis"
Biotechnol. Lett. 2000 Volume 22, Issue 18 Pages 1453-1457
M.P. Nandakumar, R. Nandakumar, B. Mattiasson

Abstract: A flow injection system for the on-line detection of the intracellular enzyme lactate dehydrogenase (LDH) during fermentation has been developed. The system is comprised of an on-line cell disintegration part, an immobilized dye based expanded bed column for the affinity capture of LDH and a fluorimetric detection unit. The system with a linearity of 0.1-5.4 U LDH mL-1 was applied for the detection of intracellular accumulation of LDH during Lactococcus lactis subsp. lactis cultivation.
Enzyme

"Integrated Flow Injection Processing For On-line Quantification Of Plasmid DNA During Cultivation Of E-coli"
Biotechnol. Bioeng. 2001 Volume 73, Issue 5 Pages 406-411
M. P. Nandakumar, E. Nordberg Karlsson, B. Mattiasson

Abstract: An integrated flow injection processing (FIP) system for the quantification of plasmids during cultivation is described. The system performs on-line sampling, cell lysis, and quantification of plasmids in an integrated manner during cultivation of E. coli. The system was operated by using a miniaturized expanded-bed column which can be used for handling samples containing cells and cell debris without interfering with the binding analysis. Two types of detectors (one measuring UV absorbance at 254 nm and a fluorometer) are used for online plasmid detection. The system was developed using standard solutions and it was successfully applied in monitoring plasmid contents during a cultivation of E. coli.

"On-line Calibration Of A Computerized Biosensor System For Continuous Measurements Of Glucose And Lactate"
Biotechnol. Bioeng. 1995 Volume 45, Issue 2 Pages 122-128
Marika Kyröläinen, Håkan Håkanson, Bo Mattiasson*

Abstract: An on-line calibration procedure for application in continuous monitoring systems has been developed. Control of the calibration value and recalibration on-line during monitoring is possible without having to disrupt the sample withdrawal. The calibration procedure has been applied and evaluated in a continuous biosensor system based on the detection of oxygen depletion during enzymatic substrate conversion by immobilized oxidases. Evaluation included on-line calibration during continuous measurements of glucose and lactate in bovine blood samples. Calibration of the complete system consisting of a sampling device, a sample handling step, a biocatalytic step, a detection step, and a data processing unit is performed by the on-line addition of a calibration solution to a blank sample which is fed through the system. The calibration cycle is completed within 5.5 min. When recalibration is carried out during monitoring, the calibration solution is added to the sample, instead of to a blank sample, and the increase in outlet singl is registered. The major advantage of this internal standard principle is that the calibration solution is fed through the whole system according to the same path as the sample solution and thus takes into account all parameters influencing the sample.

"Superporous Agarose Monoliths As Mini-reactors In Flow Injection Systems"
Bioseparation 2000 Volume 9, Issue 4 Pages 193-202
Madayi P. Nandakumar1, Eva Pålsson2, Per-Erik Gustavsson2, Per-Olof Larsson2 and Bo Mattiasson

Abstract: A new type of agarose material, superporous agarose, was used as a support material in an analytical system designed for monitoring of bioprocesses with respect to metabolites and intracellular enzymes. The superporous agarose was used in the form of miniaturised gel plug columns (15 x 5.0 mM I.D. monolithic gel bed). The gel plugs were designed to have one set of very large pores (about 50 ?m in diameter) through which cells, cell debris and other particulate contaminants from the bioreactor could easily pass. The material also had normal diffusion pores (300 A?) characteristic of all agarose materials, providing ample surface for covalent attachment of antibodies and enzymes used in the analytical sequence. The superporous agarose gel plug columns were characterized with respect to flow properties and handling of heavy cell loads as well as dispersion of injected samples (a Bodenstein number of about 40 was observed with acetone tracer at a flow rate of 1 mL min-1). To evaluate the practical performance of the superporous gel plug columns, two applications were studied: (1) on-line determination of glucose in cultivation broth (gel plug with immobilized glucose oxidase) and (2) immunochemical quantification of intracellular ?-galactosidase in E. coli (gel plug with lysozyme to achieve cell lysis and gel plug with antibodies against ?-galactosidase).
Glucose

"Flow Injection Analysis Of Intracellular β-galactosidase In Escherichia Coli Cultivations, Using An Online System Including Cell Disruption, Debris Separation And Immunochemical Quantification"
Bioseparation 1999 Volume 8, Issue 1-5 Pages 255-267
Anita Tocaj, M.P. Nandakumar, O. Holst, B. Mattiasson

Abstract: A continuous integrated process for online quantification of intracellular components has been developed. By applying the concept of expanded micro-beds in a flow injection system it was possible to first perform online cell disintegration followed by an online binding assay for quantification of a reporter protein (β-galactosidase) from the cell interior. The disintegration process involved the use of an expanded bed with immobilized lysozyme followed by ultrasonic treatment in a flow-through cell. The cell debris does not interfere in the binding assay as it is carried out in an expanded bed. The time for an assay cycle is at present approximately 35 min. This integrated system can be used for quantification of proteins down to at least 10^-7 mol/L.
Calibration

"Use Of A Micro-expanded Bed Containing Immobilised Lysozyme For Cell Disruption In Flow Injection Analysis"
Bioseparation 1999 Volume 8, Issue 1-5 Pages 247-54
M. P. Nandakumar, Anita Tocaj, B. Mattiasson

Abstract: A method for cell disruption in Flow Injection Analysis (FIA) systems has been developed. The principle involves on-line cell disruption by means of immobilized lysozyme followed by an ultrasonic treatment. In order to avoid flow problems in the analytical system, the lysozyme was immobilized to Streamline that was used in an expanded bed in the flow system. Samples of suspensions of Micrococcus lysodeikticus were treated and the success of the treatment was evaluated in terms of released protein and as a decrease in the optical density at 450 nm. The new technology offers a powerful tool in flow injection analyzes for quantification of intracellular compounds. The concept of integration, i.e. combining cell disruption with handling of cell debris and assay procedure in one continuous flow process facilitates its use and increases the probability of reaching reproducible and reliable results.

"Binding Assays In Heterogeneous Media Using A Flow Injection System With An Expanded Micro-bed Adsorption Column"
Bioseparation 1999 Volume 8, Issue 1-5 Pages 237-45
Bo Mattiasson, M. P. Nandakumar

Abstract: Competitive binding assays have been performed in flow injection systems. To further increase the versatility of the system, and to enable it to deal with samples containing particulate matter, the adsorption step was designed as an expanded bed column. Immunochemical quantification of human serum albumin was chosen as a model system to use for the development of the technology. A competitive ELISA was set up using peroxidase labelled HSA as competing ligand. The introduction of the expanded bed immunosorption column made the system tolerant to samples containing suspended particulate matter. The analytical outcome is very similar to that from the packed bed system even though more time is required for each assay cycle. The capability of the system was tested by addition of increasing amounts of yeast cells. The results clearly indicate that the system is suitable e.g. for process monitoring of fermentations.
Adsorption Calibration

"Online Monitoring Of Glucose And/or Lactate In A Fermentation Process Using An Expanded Micro-bed Flow Injection Analyser"
Bioseparation 1999 Volume 8, Issue 1-5 Pages 229-35
M.P. Nandakumar, Arvind M. Lali, B. Mattiasson

Abstract: A novel flow injection biosensor system for monitoring fermentation processes has been developed using an expanded µbed 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 analyze samples directly from a fermenter 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 fermenter, with more ease than possible with other techniques. Glucose oxidase immobilized 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 online expanded µbed FIA it was possible to follow the fermentation with Lactobacillus casei.
Glucose

"Continuous Measurements Of A Binding Reaction Using A Capacitive Biosensor"
Biosens. Bioelectron. 2005 Volume 21, Issue 1 Pages 41-48
Martin Hedström, Igor Yu. Galaev and Bo Mattiasson

Abstract: A capacitive biosensor with polyclonal antibodies raised against human serum albumin (HSA) immobilized on a gold transducer has been developed for continuous measurement of HSA in the µM-range. A mathematical model has been refined to describe integral HSA-binding curves assuming that (i) binding is essentially irreversible under the conditions used, (ii) the signal is scaled as the number of non-occupied binding sites and (iii) the rate of disappearance of available binding sites is scaled as the number of available binding sites and analyte concentration in solution. Deconvolution of the curves using the mathematical model indicates clearly that it is possible to retrieve concentration profiles (isocratic, linearly or exponentially increasing gradients) of the analyte in the continuous sample flow from the normalized integral binding (NIB) curves. The data presented constitutes the theoretical background and the first step towards the development of an analytical system allowing on-line detection of the concentration profile of the analyte from NIB-curves. Since the system can be used for extended time periods between regeneration steps, a low frequency of regeneration steps can be expected.

"Monitoring Of α-ketoglutarate In A Fermentation Process Using Expanded Bed Enzyme Reactors"
Biosens. Bioelectron. 2001 Volume 16, Issue 9-12 Pages 765-771
A. Collins, M. P. Nandakumar, E. Csöregi and B. Mattiasson

Abstract: A bienzyme flow injection system is presented for the monitoring of α-ketoglutarate produced in a fermentation process, using glutamate dehydrogenase (GDH) and glutamate oxidase (GlOx) immobilized in two serially connected expanded bed reactors. The use of expanded bed resulted in unhindered passage of the bacterial cells through the columns, and thereby the need of a separate filtering step (e.g. microdialysis) was avoided. In the first reactor, α-ketoglutarate was converted to L-glutamate by GDH in the presence of ammonia and NADH. In the following reactor, L-glutamate was converted by GlOx to α-ketoglutarate, ammonia and hydrogen peroxide, which was detected in an electrochemical flow-through cell at +650 mV vs. Pt/(0.1 M KCl). The detection limit of α-ketoglutarate in the coupled packed bed reactors was 1 µM (defined as 3 S/N), the linear range 0-100 µM, and the sensitivity 0.80 nA/µM (R(2) 0.99). In the coupled expanded bed reactors, the detection limit of α-ketoglutarate was 7 µM (defined as 3 S/N), the linear range and the sensitivity being 0-500 µM and 0.11 nA/µM (r2 = 1.00), respectively. The response time (defined as the time between peak rise and return to baseline) was 5 min for coupled packed beds (injection of supernatant), and 12 min for coupled expanded beds (injection of sample containing cellular and particulate matter). Several other parameters, such as reactor stability, flow rate dependency, bed expansion, glutamate interference, etc. were investigated and characterized. When analyzing real samples from a fermentation broth, the same results were obtained independent of the nature of the reactor system (packed or expanded bed). The hereby described system can easily be automatised and controlled from a personal computer.

"Quantification Of Nisin In Flow Injection Immunoassay Systems"
Biosens. Bioelectron. 2000 Volume 15, Issue 5-6 Pages 241-247
R. Nandakumar, M. P. Nandakumar and B. Mattiasson

Abstract: A monoclonal-antibody-based, sequential competitive-flow injection immunoassay system in expanded-bed mode has been developed for the determination of nisin. The system allows the determination of nisin in the presence of suspended particles without any significant interference, illustrating its potential for on-line monitoring of fermentation processes or the analysis of food matrices. The dose response range of the system when operated in expanded-bed mode was 6-90 µM. The detection limit under packed-bed conditions was 3 µM. The results correlated well with the results from conventional ELISA in the analysis of samples of processed cheese. When milk samples, fermentation samples and buffer were spiked with nisin, the mean recoveries were 86% for milk samples, 96% for fermentation samples and 98% for buffer solution.

"Immobilised Activated Sludge Based Biosensor For Biochemical Oxygen Demand Measurement"
Biosens. Bioelectron. 2000 Volume 14, Issue 12 Pages 883-893
Jing Liu, Lovisa Björnsson and Bo Mattiasson

Abstract: A biochemical oxygen demand (BOD) sensor, based on an immobilized mixed culture of microorganisms in combination with a dissolved oxygen electrode, has been developed for the purpose of on-line monitoring of the biological treatment process for waste and wastewater. The sensor was designed for easy replacement of the biomembrane, thereby making it suitable for short-term use. The drawbacks of activated sludge based sensor, such as short sensor lifetime, were thereby circumvented. The sensor BOD measurements were carried out in the kinetic mode using a flow injection system, resulting in 25 s for one measurement followed by 4-8 min recovery time. Based on the results of normalised sensor responses, the OECD synthetic wastewater was considered to be a more suitable calibration solution in comparison with the GGA solution. Good agreement was achieved between the results of the sensor BOD measurement and those obtained from BOD, analysis of a wastewater sample from a food-processing factory. Reproducibility of responses using one sensor was below±5.6% standard deviation. Reproducibility of responses using different sensors was within acceptable bias limits, viz.±15% standard deviation.

"A Microbial Biosensor Based On Yarrowia Lipolytica For The Off-line Determination Of Middle-chain Alkanes"
Biosens. Bioelectron. 1999 Volume 14, Issue 8-9 Pages 723-727
M. Alkasrawi, R. Nandakumar, R. Margesin, F. Schinner and B. Mattiasson

Abstract: A microbial biosensor based on immobilized psychrotrophic yeast Yarrowia lipolytica integrated to FIA for the determination of middle chain alkanes was developed. The system responded very well to middle chain alkanes even at low operational temperatures down to +5°C. The maximum sensitivity was obtained at 15°C. A linear relationship was observed between the sensor response and dodecane concentration up to 100 µM.
Temperature

"Microbial Biosensor For The Analysis Of 2,4‐Dichlorophenol"
Anal. Lett. 2005 Volume 38, Issue 7 Pages 1071-1083
Jongjit Jantra, Hamid Zilouei, Jing Liu, Benoit Guieysse, Panote Thavarungkul, Proespichaya Kanatharana, Bo Mattiasson

Abstract: A flow injection cell-based biosensor was constructed for 2,4-dichlorophenol (DCP) analysis by using a Clark-type oxygen electrode as a transducer. A mixed bacterial culture capable to biodegrade DCP was immobilized between a Teflon membrane and a dialysis membrane and attached to the oxygen electrode. Optimization of the flow rate, the injection volume, the carrier buffer concentration, and pH was carried out. Under optimum conditions (100 mM phosphate buffer, pH 7.50; flow rate 0.10 mL min-1; sample volume 100 µL), the sensor response was linear between 0.01 and 0.30 mM DCP. The detection limit was 0.02 mM DCP, and the sensor was quite stable during 5 days of operation.
2,4-Dichlorophenol Electrode Electrode Sensor Detector Optimization

"A Microbial Biosensor Using Pseudomonas Putida Cells Immobilised In An Expanded Bed Reactor For The Online Monitoring Of Phenolic Compounds"
Anal. Lett. 1999 Volume 32, Issue 12 Pages 2379-2393
Renu Nandakumar; Bo Mattiasson

Abstract: A cell based biosensor for phenolic substances has been developed. The set up is based on a flow injection system with an expanded bed column with immobilized Pseudomonas cells. The cells were immobilized on glass particles pretreated with poly (ethylene diamine). The system responds to a range of phenolic substances. Storage and operational stabilities are good. The expanded bed concept makes the system reliable also when treating samples with particulate matter.

"Sequential Injection Analysis Of Ethanol Using Immobilized Alcohol Dehydrogenase"
Anal. Lett. 1996 Volume 29, Issue 7 Pages 1109-1124
Hedenfalk, M.;Mattiasson, B.

Abstract: A sequential injection analysis (SIA) system (schematic of manifold is given) was developed for the determination of ethanol in fermentation broth. The system was optimized in terms of pH, cofactor concentration in the reagent segments and stopping time using the aspiration sequence: 1-6 mL wash/carrier buffer, NAD+ in buffer (0.15 ml), sample or standard (15 ml) NAD+ in buffer (0.15 ml) and wash/carrier buffer (0.14 ml). The optimum pH was 8.8 but the type of buffer, i.e. Tris hydrochloride or glycine/NaOH was unimportant. The stopping time had little effect reaching a plateau after 30 s. The optimum concentration of cofactor was 0.5 µmol cofactor/assay. The assay cycle was complete within 140 s. The SIA system was based on the use of immobilized NAD+ dependent alcohol dehydrogenase and the calibration range (non-linear) extended from 0.25-100 mM ethanol. The RSD (n = 50) was 1.4% at the 2 mM ethanol level.
Ethanol Fermentation broth Sequential injection Immobilized enzyme

"Online Monitoring Of D-lactic Acid During A Fermentation Process Using Immobilized D-lactate Dehydrogenase In A Sequential Injection Analysis System"
Anal. Chim. Acta 1995 Volume 300, Issue 1-3 Pages 277-285
Hun-Chi Shu, Håkan Håkanson and Bo Mattiasson*

Abstract: A PC-automated sequential injection analysis (SIA) system was used to monitor the production of D-lactic acid during the batch fermentation of Lactobacillus delbrueckii ATCC 9649. Sampling was carried out from the flux of a cellulose acetate membrane filtration unit through which the broth was continuously circulated. Cell-free permeate was diluted with 0.1 M phosphate buffer of pH 7 and passed through rotary and three-way valves to enzyme and blank reactors. The enzyme reactor contained D-lactate dehydrogenase co-immobilized with L-alanine aminotransferase on porous glass. The measuring conditions were: 0.1 M glycylglycine buffer including 70 mM glutamic acid as carrier buffer of pH 10, 4 mM NAD+ cofactor in glycylglycine buffer solution and an incubation time of 90 s. Detection was by spectrophotometric absorbance measurement (340 nm for NADH). The total analysis time was 182 s including the incubation time while the flow was stopped. The calibration graph was linear in the range 2-25 mM D-lactic acid and the detection limit was 1 mM. The results from SIA were comparable with those obtained by the offline Boehringer enzymatic kit method.
d-Lactic acid Fermentation broth Spectrophotometry Sequential injection Stopped-flow Automation Computer Method comparison Immobilized enzyme Glass

"D-Lactic Acid In Pork As A Freshness Indicator Monitored By Immobilized D-lactate Dehydrogenase Using Sequential Injection Analysis"
Anal. Chim. Acta 1993 Volume 283, Issue 2 Pages 727-737
Hun-Chi Shu, Håkan Håkanson and Bo Mattiasson

Abstract: Ground pork, homogenized with 1 M HClO4 for 10 min was diluted with water, the pH was adjusted with 2 M KOH to 10^-11 and the solution was further diluted with water. D-Lactic acid (I) was determined in the solution by a fully computerized sequential injection analysis system. A dual-piston, sinusoidal flow pump was used to aspirate the following liquid segments into the holding tube: (i) 0.1 M glycylglycine buffer containing 0.07 M glutamate of pH 10 (buffer A); (ii) 4 mM NAD+ solution; (iii) sample solution; (iv) 4 mM NAD+ solution; and (v) buffer A. The direction of the piston was reversed and v, iv and iii were transported to the enzyme column (3 cm x 4 mm i.d.) containing D-lactate dehydrogenase and L-alanine aminotransferase immobilized onto silica beads. The flow was stopped for 90 s. The piston was moved forward to expel all the reagent and products through the detector cell (30 µL) where the absorbance of NADH was measured at 340 nm. The calibration graph was linear up to 10 mM I and the detection limit was 0.1 mM. The RSD (n = 5) was 3%. The method was used to measure the change in I concentration in pork during vacuum- and chilled-storage.
d-Lactic acid Meat Spectrophotometry Sample preparation Sequential injection Immobilized enzyme Silica Stopped-flow

"Flow Injection ELISA For Process Monitoring And Control"
Anal. Chim. Acta 1991 Volume 249, Issue 1 Pages 163-168
Mats Nilsson, Håkan Håkanson and Bo Mattiasson

Abstract: A fully automated competitive flow injection ELISA is described (with diagrams). The flow system is computer-controlled and its operation involves mixing of sample and labelled antigen solution, passing of this mixture through a column containing immobilized antibodies, application of substrate solution at the column, spectrophotometric detection and column reconditioning with buffer solution The system was evaluated with the determination of IgG with a protein A - sepharose column and horse-radish peroxidase-labelled rabbit anti-human IgG.
Immunoglobulin G Spectrophotometry Automation Buffer Column Computer Process monitoring Process control

"A Chemically Sterilizable Barrier For The Protection Of Fermentors During Automatic Sampling Into A Flow Injection System"
Anal. Chim. Acta 1989 Volume 216, Issue 1 Pages 299-306
Roger Appelqvist and Gillis Johansson, Olle Holst and Bo Mattiasson

Abstract: Two three-way valves are connected by a short stainless steel tube and attached to a model fermenter. A sample can be drawn straight through the valves and into an analyzer for monitoring of oxygen or glucose. The valves are switched to an external solution after the sample has been taken. Solutions of 5% formaldehyde provide a barrier to the intrusion of micro-organisms into the fermenter, whereas water solutions are ineffective. With water in the barrier, infections occur after a few days when the system is provoked by a growing culture of E. coli at the outlet. The concept of a chemically sterilizable barrier is particularly well suited for the intermittent needs of flow-injection systems used for process monitoring.
Automation

"Flow Injection Binding Assays: A Way To Increase The Speed In Binding Analyses"
Anal. Biochem. 1989 Volume 181, Issue 2 Pages 379-382
Bo Mattiasson, Per Berdén and Torbjörn G. I. Ling

Abstract: A flow injection binding assay for glucosides and mannosides is described. The sample was mixed with enzyme label (horseradish peroxidase) and passed through a column of immobilized concanavalin A. Bound activity was measured by passing substrate (to peroxidase) and chromogenic reagent through the column and monitoring the effluent spectrophotometrically. To speed up the assay, the sample was applied in pulses and the substrate was included in the column regeneration buffer. Calibration graphs for glucosides and mannosides were rectilinear for 0.02 to 2 mM.
Carbohydrates Glycoproteins Glycosides Mannosides Spectrophotometry Enzyme Column Immobilized reagent Buffer Injection technique Chromogenic reagent

"Monitoring Of Low Concentrations Of Glucose In Fermentation Broth"
Appl. Microbiol. Biotechnol. 1999 Volume 52, Issue 4 Pages 502-507
M. P. Nandakumar, A. Sapre, A. Lali, B. Mattiasson

Abstract: 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-catalyzed reaction and subsequent spectrophotometric detection of the colored product formed. The sensor system was characterized 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.
Glucose

"Immunochemically Based Assays For Process Control"
Adv. Biochem. Eng. Biotechnol. 1992 Volume 46, Issue 1 Pages 81-102
Mattiasson B, Hakanson H.

Abstract: Immunoanalysis for process control is reviewed. The development of non-equilibrium immunoassays from a manually operated flow injection system to a completely computerized system for sample analysis as well as calibration and data evaluation is discussed. Several other approaches in using immunochemistry in biosensors for process control are discussed. The fact that reuse of antibodies raises specific demands on their properties is discussed. Even if the area is young, much progress has been achieved and one can foresee an interesting future development of the area.
Immunoassay Sensor Biotechnology Process control Review Computer