Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
2000-08-25
2003-07-22
Nguyen, Nam (Department: 1743)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C204S403010, C204S403060, C204S431000, C204S416000
Reexamination Certificate
active
06596154
ABSTRACT:
TECHNICAL FIELD OF INVENTION
The invention relates to electro-chemical measuring equipment and to a method for measuring the concentration of electrically charged particles in a liquid. The invention in particular relates to a method and an apparatus (a micro-sensor) by means of which it is possible to determine the above mentioned concentration by measuring the concentration of a secondary substance, the concentration of which is proportional to the concentration of the said first mentioned particles (primary substance), as appears from the preamble of claim
1
.
BACKGROUND OF THE INVENTION
A NO
3
−
microsensor based on immobilized nitrate reductase is described by E. Willner, E. Katz and N. Lapidot, 1992, “Bioelectroanalyzed reduction of nitrate utilizing polythiophene bipyridinium enzyme electrodes”, Bioelectrochem. Bioenerg., 29, pp. 29-45. Here a negatively charged gold cathode receives electrons that originates from the reduction of NO
3
−
to NO
2
−
, and the electrical connection between the cathode and enzymes are obtained through redox-active copolymers. The electron current from the cathode to a reference electrode was shown to be proportional with the NO
3
−
concentration in a buffer, and the electrode might be used to obtain a quantitative determination of NO
3
−
in aqueous solutions. However, it is not clear, whether this biosensor can work in natural surroundings with a variable chemical composition.
The international patent application PCT/DK96/00488 describes a microsensor primarily for on-line measurement of the methane concentration based on a bacterial conversion of methane when consuming oxygen, and measurement of the oxygen concentration. The invention enables measurement of the methane-concentration with a considerable spacious solution even in areas where steep concentration gradients occur. However, it is not possible to control the sensitivity during measurement of the methane concentration according to the invention.
The Japanese patent application No. 55072853 A describes a microsensor for measuring the concentration of ammonia in a given solution by means of bacterial conversion of ammonia with oxygen. The sensor consists of a thin bacteria layer placed on a suitable supporting element (a screen, filter a.o.) followed by a sensor for measurement of the oxygen concentration which in itself is a known technique. Also in this case, it is not possible to perform a calibration of the sensor sensitivity.
Contrary to the above systems U.S. Pat. No. 5,234,566 describes a biosensor consisting of a lipid diaphragm having at least one controlled ion channel, the electrical conductivity of which is dependent on the electrical potential difference over the diaphragm.
In principle it is, therefore, possible to calibrate this sensor by varying the applied potential difference. In this system there is no bacterial metabolism of a primary substance (the concentration of which is to be determined) to a secondary substance (the concentration of which can be measured by known techniques), as the patent only describes the conditions around the above mentioned diaphragm.
U.S. Pat. No. 5,474,660 describes a sensor for measurement of the ammonium ion concentration in a solution where the above mentioned sensor functions by measuring the concentration of ammonia. The conversion of ammonium ions into gaseous ammonia takes place by means of an electro-chemical generator having an electrode system which produces hydroxides in the area outside the sensor tip. This production of hydroixid ions is controlled by means of the above mentioned electrode system. There is no microbial participation in the measurement.
SUMMARY OF THE INVENTION
Microsensors have contributed with considerable knowledge about the decomposition of nitrogen in nature, but particularly NO
3
−
sensors have caused problems due to noise and unstability on the output signal, which is described by K. Jensen, N. P. Revsbech and L. P. Nielsen, 1993, “Microscale distribution of nitrofication activity in sediment determined with a shielded microsensor for nitrate”, Appl. Environ. Microbiol. 59, pp. 3287-3296.
The present invention combines measurement of the concentration of a primary substance through bacterial metabolism to a secondary substance the concentration of which can be measured in a known way by means of a measuring electrode, with the ability of controlling (calibrating) from the outside the sensitivity when measuring the concentration of the primary substance. The present invention in particular—but not exclusively—describes a microsensor determined for an on-line measurement of the concentration of nitrate ions NO
3
−
at bacterial consumption of these whereby laughing gas (NO
2
−
) is being produced. Measurement of the concentration of laughing gas is carried out according to a known technique, and it can be shown (see L. H. Larsen, T. Kjær, N. P. Revsbech (1997), “A Microscale NO
3
−
-Biosensor for Environmental Applications”, Analytical Chemistry, Vol. 96, No. 17, p. 3527-3531) that within a large measuring area proportionality exists between the concentration of nitrate ions and the concentration of laughing gas.
It is thus the aim of the invention to provide a method and at least one concrete microsensor that makes use of this method, in order to measure on-line continuously the concentration of a primary substance with electrically charged particles in a solution, in a way that the sensitivity of the above mentioned measurement can be varied whereby a more simple calibration of the microsensor can be carried out.
The invention essentially consists of the following three fundamental elements: 1) a measuring electrode to measure the concentration of a secondary substance in a known way where the above mentioned substance in the described embodiment is laughing gas (N
2
O
−
), 2) an area, hereinafter called “reaction chamber”, containing bacteria that brings about a metabolism of a primary substance, the concentration of which is to be determined, to the above mentioned secondary substance, and where the above mentioned reaction chamber is placed on the boundary surface between the above mentioned measuring electrode and the medium containing the primary substance, the concentration of which is to be determined, and 3) a suitable electrode means capable of applying an electrical potential difference between the substance contained in the above mentioned reaction chamber and above mentioned surrounding medium, containing the particles, the concentration of which is to be determined, in such a way that through variation of the above mentioned electrical potential difference it will become possible to vary the sensitivity of the measurement of the above mentioned primary substance.
The advantages of the above mentioned invention are that it is possible to vary externally the sensitivity of the measurement of the concentration of the primary substance, hereby being able to adapt the sensitivity to the conditions that rule in the actual measuring situation. In particular one advantage is obtained, namely that in polarization of the potential in the reaction chamber with the same charge sign as the electrical charge of the particles to be measured, it is possible to determine the zero or idle current of the microsensor. Further, by applying to the reaction chamber a large electrical potential difference relative to the surrounding medium but with opposite charge sign relative to the electrically charged particles, the concentration of which is to be determined, it is possible to determine the maximum signal amplitude of the microsensor, that is exactly the concentration of the primary substance, whereafter there will no longer exist proportionality between the concentration of the primary and the secondary substance. A calibration curve for the sensor can thus be obtained by means of just one solution of the primary substance, the concentration of which has been determined by means of another method. In general the invention can be summarize
Kjær Thomas
Larsen Lars Hauer
Barneys & Thornburg
Nguyen Nam
Noguerola Alex
Unisense Aps.
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