Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
1999-05-07
2001-05-01
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S287500, C435S288700, C435S808000, C435S817000, C204S403060, C204S415000
Reexamination Certificate
active
06225108
ABSTRACT:
The present invention relates to a microsensor for measuring the concentration of a primary medium in an environment, e.g. a liquid, gas or matrix, which microsensor has a transducer and a reaction chamber with an opening. This microsensor is of a type, which has a a casing or container around the transducer. The microsensor is provided with an opening, and the tip of the transducer is placed at a certain distance from this opening. Between the opening and the tip, a reaction chamber is formed. Furthermore, the invention relates to the application of such microsensor.
According to the invention, the microsensor is primarily intended to measure the presence and concentration of nitrate, NO
3
−
. In the following, nitrate is also called the primary medium, the presence and concentration of which is to be determined. This determination is of interest in connection with the analysis of the conversion of nitrate in agricultural cultivation. Also, the nitrate concentration is of interest when determining the degree of pollution of surface water, groundwater and waste water. However, the inventive microsensor can also be used to measure the presence and concentration of other substances, like sulphate, SO
4
2−
.
“Applied and Environmental Microbiology”, Apr. 1996, page 1248-1251, describes a microsensor which can measure the presence and concentration of nitrate. Here, the primary medium is nitrate, and measurement of its concentration is done indirectly through measurement of a concentration of a secondary medium which is nitrous oxide, N
2
O. Nitrous oxide develops as the secondary medium after reduction of the primary medium nitrate, NO
3
−
and the reduction is carried out due to denitrifying bacteria.
The known microsensor has a transducer with a tip. A housing or casing surrounds the transducer, and has an opening. Between the opening of the container and the tip of the transducer a reaction chamber whith bacteria is located. The bacteria are immobilized in the reaction chamber by means of an alginate matrix. The casing is fixed to the transducer by means of wax that is placed between the transducer and the container.
This known microsensor suffers from some disadvantages. Measurements with the known microsensor can only be performed in environments having nutrients for the bacteria, or their activity will cease. Nutrition of the bacteria in environments devoid of dissolved nutrients in this prior art reference is based on granules of glycogen or polyhydroxybutyrate contained within the bacteria used. However, these granules are exhausted after about 1-2 hours without an added electron donor.
The range of measurement and the response time for the known sensor are linearly proportional. Hence this sensor cannot be used for measurements in environments with NO
3
−
concentration exceeding 500 &mgr;M. Furthermore, it is not at all possible to increase the relation between the NO
3
−
concentration in the measuring environment and the N
2
O concentration at the transducer, and, consequently, it is not possible to have the measuring range of the microsensor exceed 5-500 &mgr;M NO
3
−
. Therefore, the known microsensor is unfit for measuring the presence and the concentration of e.g. nitrate in natural environments.
Other known sensors are the ion-exchanger based sensors which are intended for measuring nitrate in a liquid. However, this type of sensor suffers from one essential disadvantage. Its sensitivity to interference from other ions like cloride, C
1
−
and hydrogen carbonate, HCO
3
−
, contributes to errors in measurements performed with this type of sensor. This known sensor is, therefore, unfit for measurements in e.g. sea water and alcaline water like groundwater or surface water. Furthermore, the sensitivity towards HCO
3
−
interference means, that the ion-exchanger based sensors suffers from pronounced interference in biological very active environments with extensive production of HCO
3
−
such as waste water treatment plants.
From the Japanese patent application JP 60117143 a design of a sensor is known, in which enzymes are fed from a separate, external reservoir through a tube down to an immobilized enzyme membrane. In this way, the enzymes are kept active hence prolonging the period of activity of the sensor. However, the external reservoir and the projecting tube makes this sensor larger and more vulnerable to mechanical influences compared to the inventive design with only one single, outer casing. Further, this sensor is not a self contained unit; supply of enzymes comes from a external source with the function of replenishing.
The aim of the present invention is to provide a sensor, which does not suffer from the known disadvantages, and by means of which the measurement of the concentration of a primary medium, i.e. nitrate NO
3
−
, will become more precise and results in less sensitivity against interference from other substances.
Another aim of the invention is to provide a sensor using the principle of metabolism through bacteria, which sensor has an extended life span, is handy and robust and does not need manual replenishment of the bacteria or their nutrients.
As described in claim
1
, these aims are reached by using af microsensor of the type using bacteria for transforming a primary medium into a secondary medium and detecting the secondary medium as an indirect measure of the primary medium, whereby the bacteria are confined in a reaction chamber which is surrounded by an outer casing. The microsensor is characterised in that the casing delimits a chamber or reservoir containing nutrients, and thus confines the nutrients which rests against the casing, preferably directly, and that a passage is located between reaction chamber and reservoir. Through this passage nutrients diffuse from the reservoir to the reaction chamber, to be consumed by the bacteria, and through which passage also secondary medium diffuses away, i.e. in a direction opposite to the direction of the nutrient.
The invention thus solves the problem of nutrition of the denitrifying bacteria placed in the reaction chamber. By providing a reservoir with nutrients confined by the casing and a passage leading from the reservoir to the reaction chamber, a self contained regenerative sensor is achieved. The reservoir functions as an internal constant diffusive source of nutrients which regenerates the bacteria by allowing continous growth in the tip of the sensor. The reaction chamber thus functions as a true micro chemostat. Preferably, according to claim
2
, the casing surrounds both reservoir and reaction chamber whereby a practical, self cointained entity can be manufactured. As described in claim
3
, the passage is preferably located between the transducer and the casing, which results in a narrow channel through which nutrients are fed. As mentionend in claim
4
, the nutrient placed in the reservoir should contain either an electron acceptor or donor dependent upon whether the primary medium is oxidized or reduced by the the biologically active substance in the reaction chamber.
As further stated in claim
5
, the passage can contain means for influencing the net flux of molecules. In a situation with varying concentration of the primary medium, it is important to guide or influence the diffusion. A sudden decrease in the concentration of the primary medium in e.g. the sea water would cause a reversal in the direction of natural diffusion of secondary medium. Instead, accumulated secondary medium would now diffuse from the reservoir to reaction chamber, resulting in incorrcet measuring. By adding certain means to the passage, this situation can be avoided.
By letting the passage end up in a reservoir in the casing behind the transducer tip, an advantage is achieved in that the passage can be used for adding nutrients to the bacteria in order to maintain the bacterial activity. In establishing a passage which stretches behind the transducer tip, essential improvements are achieved when measuring the presence and the
Kjær Thomas
Larsen Lars Hauer
Revsbech Niels Peter
Beisner William H.
Lee Mann Smith McWilliams Sweeney & Ohlson
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