Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-06-06
2004-09-07
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S034000, C204S403010
Reexamination Certificate
active
06787331
ABSTRACT:
BACKGROUND
This application claims priority to German Application No. 100 28692.5 filed Jun. 9, 2000.
The invention concerns a process for examining of membrane enclosed biocompartments, wherein the biocompartments are arranged in amicro-flow chamber. This micro-flow chamber is subject to the through-flow of a culture medium containing an active agent and which is in contact with the said biocompartments and possibly has an effect thereon. Further, the pH value of the culture medium present in the micro-flow chamber can be measured. In this arrangement, the biocompartments can hold, for instance, biological cells, microorganisms (fungi, bacteria), biochemical compartments and/or mitochondria.
EP 0 394 406 B1 discloses a process of the type known in the art, in which living, biological cells are placed in a micro-flow chamber being subjected to the through-flow of a culture medium and which chamber has a silicon sensor on its bottom. A reference electrode is provided in the micro-flow chamber, which is in continual contact with the culture medium. In the case of this previously known process, in a first process step, the through-flow of the culture medium is stopped in the micro-flow chamber. Under these circumstances, the pH value, serving as a metabolic indicator of substance change in the culture medium residing in the micro-flow chamber, is determined by measurement of the electrical potential between the silicon-sensor and a reference electrode. With this process, it is possible to measure, for instance, the action of a cell-affecting agent on the metabolism of the cells. A detriment, however, lies in the fact, that the process enables only one determination in regard to a yield of acid metabolic products, while other changes caused by the metabolism of the cells are not given consideration.
DE 44 17078 A1 makes known a process wherein biological cells in a micro-flow chamber are simultaneously examined by means of a plurality of different micro-sensors. As this is carried out, in addition to the acidification, at the same time the respiration of the cells in the culture medium is monitored, which enables a more exact investigation of the action on the cells of an agent contained in the culture medium. This procedure, however, has the disadvantage, that the measured values for pH and oxygen content are determined at different places and thus of differing biological cells present in the micro-flow chamber. Cell cultures, cell divisions, tissues and the like which are to undergo examination as biological material are, however, often heterogenous, that is, different cells can yield different signals. In this known procedure, on this account, imprecisions in the investigation of an agent in the culture medium can occur if the pH value and/or the oxygen content at the points of determination of the sensors deviate in pH and oxygen content from one another.
SUMMARY
Thus, it is the object of the invention, to make available a process for the examination of membrane enclosed biocompartments, which enables the measurement at one location of several metabolic parameters of the said biocompartments.
The achievement of this object is found especially in that:
a) the concentration of substance which is yielded or absorbed by metabolism in the micro-flow chamber in a partial increment of the culture medium in the active area of the biocompartments is indirectly measured, since between an operational electrode placed in said partial increment and a distanced reference electrode, an electrical potential is so applied to the culture medium, that hydroxyl or hydrogen ions are formed from substances in the culture medium,
b) at a measuring point in the partial increment of the culture medium during the application of the said electrical voltage, at least one first value for a pH value of the culture medium is determined,
c) subsequently, the electrical voltage will be turned off or changed in such a way, that the formation of hydroxyl ions and hydrogen ions from the said substance ceases,
d) shortly before or after the measurement of the first value at shut-off voltage, or by a voltage in which the generation of hydroxyl and hydrogen ions from the said substance is not possible, at least a second measurement value is determined for the pH value of the culture medium,
e) from the first and the second measured values, a difference is calculated,
f) the steps a) to e) are repeated at least once,
g) from a difference between at least two of these measured value differences, the concentration change of the material in the culture medium, and from the difference between at least two of the first pH measurements or the second pH values, the acidification or alkalinizations of the culture medium can be determined,
h) and from the so obtained measurement values for the concentration change as well as the acidification or alkalinization, the metabolic activity of the biocompartments can be determined.
The basis of the process lies in the recognition, that the biocompartments continually change the pH value of the culture medium at a somewhat constant increase. Further, this alteration of pH value runs essentially slower than the pH change arising from an application of electrical voltage for the generation of hydroxyl ions (OH
−
ions) or hydrogen ions (H
+
ions) from substances in the culture medium yielded by, or absorbed by metabolism in the biocompartments. This contributes to the fact that it becomes possible in spite of the superimposing of the ions of hydroxyl and/or hydrogen yielded to or taken from the culture medium by the biocompartments with those ions of hydroxyl or hydrogen from substance carried in the culture medium, at one and the same location, and with only a single pH-sensor, to measure both the substance which arises from the concentration change activated by the biocompartments of the material yielded to or taken from the biocompartments as well as the pH change in the culture medium caused by the biocompartments. With the inventive process, it becomes possible to determine the respiration of the biocompartments by the measurement of the oxygen concentration change, as well as the concentration change of nitrogen monoxide (NO), hydrogen peroxide, and/or other compounds containing oxygen and/or hydrogen in a chemically bound form, such as, for instance, molecules which are present in the cell surfaces. A dependency of change of concentration and/or pH value based on localization caused by a difference in the individual cells of the cell culture has practically no effect on the accuracy of the determination of the metabolism of the cells undergoing examination.
From Sohn, B.-H., Kim, C.-S., “A new pH-ISFET-based dissolved oxygen sensor by employing electrolysis of oxygen” in
Sensors and Actuators
B34, (1996), pages 435-440, is, to be sure, an already disclosed process, contrary to the generic type, in which the oxygen content in analytes is measured indirectly by means of the pH value, wherein the electrical potential on the analyte is so far reduced in the negative direction, that from such oxygen as is in the analyte, hydroxyl ions are formed, and wherein these hydroxyl ions are detected by means of an ion selective sensor. The analyte contains, however, no biocompartments which change during the measurement, that is, which change the analyte by means of acid metabolic products and the consumption of oxygen. Also, this known procedure results in no evaluation of the sensor signal in regard to the determination of respirational activity and acidification when potential is not reduced.
In a particularly advantageous embodiment of the process, a provision is made that the sequence comprising the steps a) to g) is run through at least twice and that in these at least duplicated operations, the polarity is differently chosen between the electrical potential applied between the operational electrode and the reference electrode. This makes it possible to measure in the culture medium, using only one pH-value sensor, both the concentration change of electro-negative substa
Gitomer Ralph
Micronas GmbH
Volpe and Koenig P.C.
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