Carl Fredrik Mandenius

Studies on guest selective molecular recognition on an octadecyl silylated silicon surface using ellipsometry

Abstract Application of the optical surface method ellipsometry to guest selective recognition is described. Detection of the affinity binding by guest recognition to an octadecylsilane derivatized silicon surface with vitamin K1 is exemplified with the technique.

The interaction of proteins and cells with affinity ligands covalently coupled to silicon surfaces as monitored by ellipsometry

Two methods for the chemical binding of biomolecules to silicon surfaces are described. The first method utilizes an alkyl silane and a nucleophilic reagent to join the biomolecule to the silicon surface; the second method involves crosslinking with glutaraldehyde in order to couple the biomolecule and albumin molecules, which have first been physically adsorbed. The course of binding to the silicon surface has been followed with the aid of ellipsometry. This optical measuring technique estimates the thicknesses of, e.g., organic layers, by measuring the polarization properties of a light beam before and after reflection at surfaces. The method by which the binding of a biomolecule to its corresponding affinity ligand on silicon wafers can be followed with this technique is reported. The systems studied are concanavalin A-Saccharomyces cerevisiae cells, immunoglobulin G-Staphylococcus aureus cells, and an NAD-analog-lactate dehydrogenase. With ellipsometry it was possible to assess how the incubation time and the concentration of the cells and the biomolecules added influenced the results. It was found that an increasing time of incubation and higher concentration resulted in a more complete coverage of the silicon wafer surfaces.

Evaluation of a dialysis probe for continuous sampling in fermentors and in complex media

Abstract A dialysis probe is described for continuous sampling from complex solutions, such as fermentation broth, milk or waste water, to yield samples suitable for liquid chromatography, flow injection analysis, enzyme calorimetry, etc. The analyte is transferred to a flow stream separated from the sample by a dialysis membrane that is protected from fouling by a strong tangential flow of the sample solution. This flow is accomplished by placing a magnetic stirring bar close to the membrane surface. The device is constructed of materials permitting the probe to be steam-sterilized when mounted inside a fermentor.

Gas phase silylation, a rapid method for preparation of high-performance liquid chromatography supports

Abstract A new method for preparing chromatographic supports is described. Porous silica was treated with gaseous silanes and gaseous triethylamine at high temperature and reduced pressure. The silylation procedure was rapid and gave supports with a covalently bound monolayer of aminopropyl-or epoxysilane. Diol silica prepared by gas phase silylation was compared with diol silica prepared in the aqueous or organic phase (toluene). Seven proteins were chromatographed and the protein recovery, plate number and peak asymmetry were calculated. Silica prepared by the gas phase silylation method showed improved performance. The separation of carbonhydrates on aminopropylsilica and the isolation of lactate dehydrogenase with nicotinamide-adenine dinucleotide-silica, prepared from gas phase silylated diol silica, are described.

Monitoring and control of enzymic sucrose hydrolysis using on-line biosensors

SummaryPreviously reported flow microcalorimeter devices for enzymic reaction heat measurement, enzyme thermistors, have here been extended with systems for on-line sample treatment. Glucose analysis was performed by intermittent flow injections of 50 μl samples through such an enzyme thermistor device containing immobilized glucose oxidase and catalase. Sucroce analysis was performed by allowing diluted samples to continuously pass through an additional enzyme thermistor containing immobilized invertase. The reaction heats were recorded as temperature changes in the order of 10–50 m°C for concentrations of 0.05–0.30 M glucose or sucrose present in the original non-diluted samples.The performance of this system was investigated by its ability to follow concentration changes obtained from a gradient mixer. The system was applied to monitoring and controlling the hydrolysis of sucrose to glucose and fructose in a plug-flow reactor with immobilized invertase. The reactor was continuously fed by a flow of scurose of up to 0.3 M (100 g/l). Glucose and remaining sucrose were monitored in the effluent of the column. By using flow rate controlled feed pumps for sucrose and diluent the influent concentration of sucrose was varied while the overall flow rate remained constant.On-line control of the effluent concentration of lucose and sucrose was achieved by a proportional and integral regulator implemented on a microcomuter. Preset concentration of glucose in the effluent could be maintained over an extended period of time espite changes in the overall capacity of the invertase reactor. Long delay times in the sensor system and the enzyme column made it necessary to carefully tune the control parameters. Changes of set-point value and temperature disturbances were used to verify accuracy of controlling performance.

Process control of an ethanol fermentation with an enzyme thermistor as a sucrose sensor

SummaryAn enzyme thermistor was used to monitor and control the sucrose concentration in a conversion of sucrose to ethanol with immobilized yeast. A continuous stirred tank reactor containing calcium alginate to entrap Saccharomyces cerevisiae was used. The enzyme thermistor was continuously measuring the sucrose concentration in the fermenter with an on-line arrangement giving stable and reproducible heat signals. The control of the sucrose concentration level was performed with an analogue PI-controller.

Detection of biospecific interactions using amplified ellipsometry.

Amplified detection of biomolecules and biological interactions using an optical surface technique, ellipsometry, is demonstrated for two biosystems--immunoglobulin G with anti-immunoglobulin G (IgG) and the lectin concanavalin A (Con A) with yeast cells. In order to improve the sensitivity of the ellipsometer signal, an amplifier conjugate is formed by binding the affinity ligand to a 12-nm silica particle which is readily detected by the ellipsometer. Thus by using conjugates of IgG-silica and Con A-silica, amplifications of five to seven times have been obtained enabling detection of less than 20 pg/mm2 of biomolecular material.

Immobilization of pyranose oxidase (Phanerochaete chrysosporium): Characterization of the enzymic properties

Immobilization of pyranose oxidase (E.C.1.1.3.10) from Phanerochaete chrysosporium is described. The enzyme was bound to a glass-beaded support according to the glutardialdehyde, diazo, and carbodiimide methods with activity yields of 10%-23.3%. Characterization of the enzyme immobilized with the glutardialdehyde showed enhanced operational, storage, and temperature stability. The temperature optimum remained unchanged, but the pH optimum was slightly altered. Kinetic properties and the relative substrate specificities for glucose and xylose showed certain differences.

Optical surface methods for detection of nucleic acid binding

Abstract Detection of DNA-binding and hybridization using pseudo-Brewster angle reflectometry and ellipsometry is described. DNA was attached to planar silicon surfaces coated with a 150 A layer of nitrocellulose or with a monolayer of aminosilane. Poly-L-lysine was used as a linker between the modified surfaces and the DNA. The course of the reassociation was monitored continuously with the reflectometry method. A reassociation time of approximately one hour was observed for polynucleotides of 0.5–1.0 kb.

ENZYME THERMISTORS FOR PROCESS CONTROL

The development of automation in fermentation control has been hampered mainly by lack of suitable sensors to monitor and control the events in the fermentor. Normally, only a few parameters are registered and decisions are made based on empirical laws concerning what is really taking place in the fermentor. During the last ten years, development of specific sensors has taken place. Specific electrodes, based on a selective electrode in combination with a preparation of immobilized enzyme, have been developed.’ So far, however, such electrodes have been used mainly in clinical analysis,’ although some effort has been made to make them applicable to fermentation control.* A quite different detection principle is utilized in the enzyme thermi~tor .~ Here, a preparation of immobilized enzyme is placed in close contact with a thermistor. When substrate is fed to the enzyme, product is formed together with the liberation of a small amount of heat. This heat effect, measured as a temperature increase by the thermistor, is used to detect and register the concentration of the substrate. These new sensors, electrodes and thermistors, have been shown to work satisfactorily in the laboratory, but their applicability to real, large-scale fermentations has yet to be demonstrated. When analyzing the events in a fermentation, usually over several days, it is often sufficient to sample intermittently, although in some cases it might be preferable to register continuously. FIGURE 1 shows a schematic drawing of the type of enzyme thermistor now used in our laboratory. A thermostated aluminum cylinder contains the heat-exchanger tubing and provides a constant temperature environment to the enzyme column (0.2-1.0 ml). There are two parallel fluid lines, which could be used either independently or with one of them as a reference system.’ The sample/buffer is pumped through the enzyme thermistor unit with a peristaltic pump at a flow rate of 0.5-2.0 ml/min. The temperature at the outlet of the column is continuously monitored by a thermistor connected to a sensitive Wheatstone bridge. At the highest amplification, the recorder output is 100 mV for a temperature change of O C .