Plastic and nonmetallic article shaping or treating: processes – Heat polishing of inorganic article surface outside of mold
Reexamination Certificate
1999-05-13
2001-11-13
Warden, Jill (Department: 1744)
Plastic and nonmetallic article shaping or treating: processes
Heat polishing of inorganic article surface outside of mold
C204S403060, C204S450000, C435S285200, C435S287100, C435S287300, C435S288300, C435S288400, C422S105000
Reexamination Certificate
active
06315940
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a microelement device having a plurality of microelements, arranged on a substrate, for making contact to cells present in a liquid, preferably biological environment.
The invention further relates to a method for making contact to cells present in a liquid, preferably biological, environment above a substrate, in which a contact is created between the cells and microelements.
Lastly, the invention relates to a method for manufacturing a microelement device having a plurality of microelectrodes, in which the microelements are arranged on a substrate.
It is known to use so-called microelectrode devices for the investigation of biological cells. The microelectrode electrodes serve in this context, for example, to stimulate the cells or to sense potentials. The investigations can be conducted in a biological environment or in an artificial environment. This can be, for example, a suspension having artificial vesicles made of lipids, pores being incorporated into the vesicle shell as a model system for biological cells. For this purpose the arrangements comprise, on a substrate, a plurality of microelectrodes whose dimensions are of approximately the order of magnitude of the cells, i.e. in the range from a few &mgr;m to several tens of &mgr;m.
German Patent Application P 195 29 371, of earlier priority but not previously published, discloses a microelectrode device of this kind.
To measure bioluminescence or chemoluminescence, e.g. as a reaction to chemical stimulus (toxins, drugs), and to measure changes in light absorption caused by such a stimulus when a light source is used above the cells, it is possible to utilize light-sensitive microelements, for example microphotodiodes, which are sensitive to specific spectral regions.
Microelectrodes, microphotodiodes, and the like are referred to globally, in the context of the present invention, as “microelements.”
The following problems, inter alia, arise with microelectrode arrangements of conventional type and with the methods carried out therewith:
When the microelement device is brought into contact with a suspension, i.e. a liquid, for example biological, environment in which cells are present, it is more or less a matter of chance whether one cell or another settles on a specific electrode. In practice, the cells can generally be caused to settle on an electrode only with partial coverage, so that stimulation of the cell or sensing of a cell potential is confined to that partial surface. When the cell is stimulated, for example, a portion of the stimulation energy is lost in the suspension which acts as the electrolyte.
In addition, the cells only rest loosely on the electrodes. This can result in problems in terms of sealing resistance with respect to the reference electrode. In addition, the contact is very sensitive and is disrupted in response to even extremely small mechanical influences, since the cells detach from the contact.
SUMMARY OF THE INVENTION
It is therefore the object of the invention to provide an improved microelement device which eliminates the problems cited above. It is a second object of the invention to disclose a method of making such a microelement device. It is a further object of the invention to provide a method for making contact to biological cells. The intention in particular is to make it possible to bring individual cells in controlled fashion from a liquid environment into contact with the microelements and to make good contact there.
These and other objects are achieved by a microelement device which comprises means for guiding and/or isolating and/or mechanically attracting the cells onto the microelements.
With respect to the method for making contact to cells, this object is achieved, according to the present invention, by the fact that a guiding force and/or attractive force is generated between the cells and the microelements or the substrate.
Lastly, in the case of the method of making such a microelement device, the object is achieved, according to the present invention, by the fact that the substrate is manufactured at least from a base plate and a cover plate located thereabove.
Two advantages result from the fact that the cells are mechanically attracted to the microelements:
As long as the cells are still located unconstrainedly in the liquid environment, the result of the attractive force is that the cells move in controlled fashion onto the elements. It is therefore no longer a matter of chance whether one or the other cell settles on a predefined microelement.
As has already been mentioned, the present invention refers to microelements, i.e. preferably microelectrodes or microphotodiodes, without being limited to this application. This also applies, in particular, to the exemplary embodiments mentioned below which discuss microelectrodes, although the statements in most cases are also applicable to microphotodiodes and similar microelements.
In addition, in the case of a microelectrode the result of a continuing attractive force is that the cells are pressed with a certain contact force against the microelectrodes, and the result thereof is a particularly good sealing resistance with respect to the reference electrode, and the adhesion is moreover also mechanically stable. The electrical resistance and thus the measurable signal (action potential) are substantially improved.
Although it is known from a different technical field (the so-called “patch-clamp technique”) to aspirate cells by negative pressure against a pipette (cf. the U.S. periodical “Nature” Vol. 260, pp. 799-801, 1976), in the patch-clamp technique the pipette must be specifically guided to one individual cell. In the patch-clamp technique, the cells to which contact is to be made are not moved, since as a rule they are adhering to the substrate. The operation of making contact to cells using patch-clamp pipettes is substantially facilitated if the cells are immobilized by adhesion. Translocalization of adhering cells almost always results in lethal cell damage. The principal disadvantage of the patch-clamp technique lies in the limitation on the number of cells to which contact can be made simultaneously, since for space reasons it is impossible to introduce an arbitrarily large number of pipettes into the culture chamber. The invention, on the other hand, has the advantage that contact can be made simultaneously to a plurality of cells without the aforementioned space problems occurring.
The use of a base plate and of a cover plate separated therefrom offers advantages, in the context of the present invention, in particular when the arrangement according to the present invention is used repeatedly. For example, the base plate and the cover plate can be reused several times, either each individually or together. In addition, different manufacturing methods and materials can be used for the two plates.
Lastly, an advantage results, in terms of shaping of the electrode geometry, from the geometry (openings) of the cover plate and a resulting simplification in manufacture.
In a preferred embodiment of the arrangement according to the present invention, the means exert a negative-pressure force on the cells.
The advantage of this feature is that the necessary attractive force can be generated by purely mechanical means, i.e. by generating a negative pressure or vacuum.
In another exemplary embodiment of the invention, the means exert a hydrodynamic force on the cells.
The advantage of this feature is that the desired force can again be generated in simple fashion by generating a flow in the liquid biological environment.
It is further preferred if the means comprise channels which open out at a contact surface of the microelements.
The advantage of this feature is that the cells can be brought to the microelements and retained there, practical centering of the cells on the microelements being possible at the same time.
In this exemplary embodiment, it is further preferred if the channels can be connected to a source of negative pressure.
The cells can th
Egert Ulrich
Nisch Wilfried
Stelzle Martin
Stett Alfred
Hamrick Claude A. S.
NMI Naturwissenschaftliches und Medizinisches Institut an der Un
Oppenheimer Wolff & Donnelly LLP
Starsiak Jr. John S.
Warden Jill
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