Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
2000-11-20
2002-10-01
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S512000, C436S513000, C436S536000, C435S006120, C435S007100, C435S007200, C422S050000, C422S051000, C422S051000, C422S063000, C422S068100, C422S082010, C422S082020, C204S194000, C204S280000, C204S286100, C204S400000, C204S403060, C204S406000, C205S545000, C205S640000
Reexamination Certificate
active
06458600
ABSTRACT:
This is the National Stage Filing of PCT/DE98/03437 filed Nov. 20, 1998 and published Jun. 3, 1999.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to a structure, on the surface of the support material of which structure molecular layers are immobilized so as to be electrically addressable, a method for the electrically addressable immobilization of molecules, a device for carrying out this method, and the use of this structure as a chemo- and/or biosensor, in particular as a multisensor system for chemical, biological, and physical assays, and for applications in the combinatorial synthesis on the boundary surface.
2. Prior Art
Both chemosensors and biosensors, i.e., comparatively small measuring arrangements, are increasingly needed to make it possible to carry out chemical or biochemical analyses rapidly and at the site of the occurrence. Compared to the immunological assay systems, they have the special advantage that they are able to quantify, preferably continuously, for example, concentrations of (bio)chemical substances within a short period of time and without a time-consuming preparation of the samples. A biosensor should therefore be small, which offers the additional advantage that it can be used in the vicinity of the site of the analysis; for example, using enzymatic biosensors, diabetics are able to determine their blood sugar level within a few minutes.
Thus, chemo- and biosensory analysis includes both detection and quantification. The process of molecular detection, for example, plays an important role in biology. For example, a cell should be able to detect a potassium ion and should not mistake it for a sodium ion which is very similar [to the potassium ion], the enzyme glucose dehydrogenase should only degrade glucose but not fructose, and the immune system should be able to recognize foreign organisms and substances that have invaded the system before it attacks them. This means that molecules that recognize the targeted substances are needed to make it possible to develop chemo- and biosensors. At the same time, the many other substances that exist side by side with the targeted ones must be ignored. Thus, a biosensor must be able to meet the following requirement, i.e., that the process of recognizing/coupling can be detected and “translated” into a measurable signal. During this “translation,” however, serious problems arise so that there is a need for a system that makes detection easy. In systems that are used in the form of monosystems, it is not necessary to immobilize the molecules since only one single species has to be recognized and identified. If, on the other hand, multiple systems are to be used, such as multisensor systems, with which different molecules are to be detected at the same time, it is necessary to ensure an addressable immobilization of the molecular layers on the surface of the support material, thereby making it possible to detect the recognition of a signal. This type of system is not known in prior art.
In prior art, several methods for an addressable immobilization are available, with which it is possible to specifically couple several different types of molecules to a surface of the support material. Yershov et al. (Yershov, G., Barksy, V., Belgovskiy, A., Kirillov, E., Kreindlin, E., Ivanov, I., Parinov, S., Guschin, D., Drobishev, A., Dubiley, S., Mirzabekov, A., Proc. Natl. Acad. Sci. 43 (1996), pp. 4913-4916) as well as Blanchard et al. (Blanchard, A. P., Kaiser, R. J., Hood, L. E., Biosens. & Bioelectron. 11 (1996), pp. 687-690) describe the use of this technique, for example, to produce DNA arrays. In addition, these techniques are used for the micromechanical addressability and for the optical addressability. U.S. Pat. No. 5,412,087 describes the optical immobilization by coupling functional groups to photosensitive protecting groups. The system is activated by cleaving off the protecting groups by means of photolytic degradation. Chrisey et al. (Chrisey, L. A., O'Ferall, E., Spargo, B. J., Dulcey, C. S., Calvert, J. M., Nucl. Acids Res. 24 (1996), pp. 3040-3047) describe a different approach according to which the adsorption is carried out using the photoresist technique.
The known immobilization methods mentioned above, however, have many drawbacks which limit their applicability. Thus, for example, the resolution of the micromechanical immobilization is limited by the size of the individual spray particles. In the above-described method used by Yershov et al. (1996) and Blanchard et al. (1996), at best an optical resolution of approximately 100 &mgr;m is possible. During the optical immobilization according to U.S. Pat. No. 5,412,087, it is always necessary to work with protecting groups which require that specific conditions (such as solvents, darkening the work area because the protecting groups are sensitive to light, etc.) be maintained and which must be removed at a certain point in time. The above-mentioned photoresist technique by Chrisey et al. (1996) is very time- and cost-consuming since a large number of photomasks must be used.
SUMMARY OF THE INVENTION
Thus, the problem to be solved by the present invention is to make available a structure which is able to both bind and detect molecules and thus to make them qualitatively and quantitatively determinable. An additional problem to be solved by the present invention is to make available a method that makes it possible for molecules to be electrically addressably immobilized or desorbed on surfaces and thus to avoid the drawbacks mentioned. In addition, yet another problem to be solved by the present invention is to design a device for carrying out the method described by this invention and thus for manufacturing the structure described by this invention.
This problem is solved by making available a structure with electrically addressable immobilized molecules, which structure comprises a support material, one and/or a plurality (1 to n) of electrically conductive support surface(s) which is/are located on this support material, and one and/or a plurality of immobilized identical and/or different receptor(s).
Although it is known that it is possible to influence the chemical adsorption of molecules on an electrode by changing the applied chemical potential and that this dependence on the potential also applies to the chemical adsorption of thiol compounds on electrodes, the work that led to the present invention focused on the conditions under which, for example, the coupling between gold and sulfur is stable. Surprisingly, however, it was discovered that a chemically adsorbed molecular layer on an electrode is stable only within a specific pH-dependent range of potential. These results go far beyond the findings that have been described in the pertinent literature by Imabyashi et al. (Imabyashi, S., Iida, M., Hobara, D., Feng, Z. Q., Niki, K., Kakiushi, T., J. Electroanal. Chem. 428 (1997), pp. 33-38).
The problem of making available a method for the manufacture of a structure of the type mentioned above is solved by addressably immobilizing molecules on surfaces in a simply manner. This method comprises the following steps:
(a) a support material with n electrodes, where n stands for an integral number, is introduced into a flow-type cell which contains the electrolyte solution and the molecules to be immobilized (receptors),
(b) either an adsorption potential or a desorption potential is applied to each separate electrode, as a result of which the uncoated electrode regions are kept inert by the desorption potential, and the adsorption potential that has already been applied to the coated electrode regions prevents the desorption of molecules as well as the adsorption of molecules that have a different structure, which ensures that the electrode region is occupied by the first type of molecules and the molecules are immobilized and securely coupled to the surface of the support material, and
(c) the adsorption of molecules on the electrode(s) is measured as a change in the signal.
The proce
Mirsky Vladimir M.
Riepl Michael
Bianco Paul D.
Fleit Martin
Fleit Kain Gibbons Gutman & Bongini
Le Long V.
Padmanabhan Kartic
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