Optics: measuring and testing – By dispersed light spectroscopy
Reexamination Certificate
2000-12-30
2002-08-13
Warden, Jill (Department: 1743)
Optics: measuring and testing
By dispersed light spectroscopy
C356S246000, C436S164000, C436S165000
Reexamination Certificate
active
06433868
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a process for the differential spectroscopical and differential fluorometric determination of the interaction of substances with the aid of a microtitration plate and to the use of a microtitration plate for the differential spectroscopical and differential fluorometric determination of the interaction of substances.
The spectrophotometric determination of substances in homogeneous solutions is important in a large number of fields, e.g., in analyses of the environment, in basic research, in medical diagnostics, in forensic studies, etc. In the course of these determinations, substances such as nucleic acids, proteins, enzymes, their substrates, etc. are determined, ie., their presence is established and/or quantified. In these cases, it is often of interest to determine the interaction of various substances with each other. Differential spectrophotometry or differential fluorometry can be used to detect the interaction of substances with each other by way of their changed spectral or fluorometric properties. The spectral or fluorometric changes are caused either by direct interaction of the substances with each other or by binding induced configuration changes.
Similarly, solvent dependent configuration changes can be detected or aggregation equilibria can be observed in dependence upon the medium and the concentrations of substances.
Differential spectrophotometric or differential fluorometric measurements are usually made with the aid of two tandem cuvettes, namely a sample cell and a reference cell. A tandem cuvette comprises two chambers separated by a vertical partition. The solution of a first substance is put with identical concentrations into one chamber of each the sample cell and the reference cell for differential spectrophotometric or differential fluorometric measurements. The two other chambers are filled with buffer solution. In the subsequent measurement, which is made with a horizontal light beam passing through both chambers, identical spectrophotometric and fluorometric properties of the two tandem cuvettes must be observed. After that, a well defined amount of a second substance is added to the solution of the first substance in the sample cell and the same amount is filled into the buffer filled chamber of the reference cell in order to compensate for ligand absorption or ligand fluorescence. In order to avoid concentration differences, identical amounts of the buffer solution must be added to the solution of the first substance in the reference cell. After that, as described above, measurements are made a second time and the readings are compared. In view of the usually low spectral and fluorometric changes, this method is very sensitive in regard to concentration differences and therefore the respective additions of the second substance must be small as far as their volume is concerned. The limit of the determinations is given by the solubility of the substance in the buffer. Finally, when the substances are mixed, care must be taken that no solution is removed from the cells.
SUMMARY OF THE INVENTION
The technological problem underlying the present invention is to provide a process which overcomes the above cited shortcomings, in particular, minimises concentration effects in order to increase the sensitivity of the measurements, and which is capable of analysing substances of low solubility and of reducing errors due to mixing, as well as of facilitating rapid, universally applicable, parallel and therefore low cost determinations of interactions of substances.
The present invention solves the technological problem by providing a process for determining the differential spectroscopical and fluorometric interaction of at least two different substances by means of a microtitration plate containing a plurality of small bowls, wherein a lid with at least one trough associated with one small bowl is associated with the microtitration plate, and wherein a first solution of a first substance is placed into a small bowl, a second solution of a second substance is placed into the trough associated with the small bowl, and a third solution containing a mixture of the first substance and the second substance is placed into a further trough and/or further small bowl so that subsequently the differential spectroscopical and/or differential fluorometric determination can be carried out by vertical irradiation through the filled trough and the small bowls with electromagnetic waves. Conventional photometers and fluorometers, ie., ELISA or MTP readers can be used for these measurements. Thus, the invention provides that two different substances are arranged separated from each other in vertically superimposed relationship in a region characterised as the reference region, whereas the mixture of the substances is situated in a sample region of the microtitration plate and can be analysed at the same time.
If the first substance interacts with the second substance, accompanied by a change in the spectral and/or fluorometric properties, the extinction coefficients or the fluorescence characteristics of at least one of the substances in the third solution changes, ie., in the mixture of the two solutions, in comparison with the reading obtained from the two separate solutions. These changes in the extinction coefficient allow conclusions about the substance interactions and the substance properties and structures. The extinction coefficients or the fluorescence characteristics are constant if there is no interaction or in null balance.
With the identical geometrical features preferred in accordance with the invention for the small bowls of the microtitration plate and the troughs in the lid, there are identical absorptions in the sample region, ie., in the region of the microtitration plate in which the trough containing the mixture of the first and the second solution is situated, and the reference region, ie., in the region of the microtitration plate in which the first and the second solution are arranged separated from each other and in superimposed relationship in trough and small bowls, provided that no solutions were filled in. If the geometrical features.of the respective small bowls or troughs are nonidentical, a geometrical factor must be introduced in the determination of the absorption.
In a particularly preferred embodiment, the invention relates to the aforementioned process, wherein the third solution is provided by mixing a solution of the first substance with a solution of the second substance, with the volume and/or substance concentrations of the two solutions used for the preparation of the third solution each being equal to those of the first and of the second solution.
In a further preferred embodiment, the invention relates to an aforementioned process, wherein the height of the third solution layer in the small bowl or the trough containing the third solution is equal to the sum of the heights of the layers of the first and the second solution in the small bowl and in the trough in which these solutions are situated.
In a further preferred embodiment. the invention provides that the heights of the filled in first, second, and/or third solutions are standardised, ie., that the development of menisci or reading errors resulting from different levels of filling of the solutions are precluded by using in accordance with the invention a lid with at least one trough, wherein the base section of this trough ensures a well defined length of the light path in the small bowl from the base portion of the small bowl to the base section of the trough arranged on top.
The present invention envisages the use of a microtitration plate containing a plurality of small bowls, with at least part of the microtitration plate covered by a lid having at least one trough associated with a small bowl. The essential features of such sample carrier systems have been described, for example, in DE 44 05 375 A1 which, as far as the production and the structure of such sample carrier systems is concerned, is included
Bernhagen Jürgen
Brunner Herwig
Vitzthum Frank
Fraunhofer-Gesellschaft zur Förderung der Angewandten Forschung
Gakh Yelena
Ostrolenk Faber Gerb & Soffen, LLP
Warden Jill
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