Biosensors with porous chromatographic membranes

Details Biosensors with porous chromatographic membranes Biosensors with porous chromatographic membranes

2001-07-13

2004-04-27

Olsen, Kaj K. (Department: 1753)

Chemistry: electrical and wave energy

Apparatus

Electrolytic

C204S403100, C204S403110, C204S403140

Reexamination Certificate

active

06726818

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to biosensors with porous membranes comprising
(a) at least one substrate;
(b) an electrode layer patterned on the substrate, consisting of an electrode system and a circuit connector;
(c) an insulator, for separating the electrode system and a circuit connector formed on parts of the electrode layer;
(d) a porous membrane covered with the surface of the electrode system by the insulator; and
(e) a protective membrane for protecting the porous membrane, formed on the porous membrane, or upper substrate containing a sample inlet for protecting the porous membrane as well as being introducible samples wherein, when a whole blood sample is introduced to the biosensor, the whole blood sample is separated into its components during the chromatographic motion through the porous membrane so that only blood plasma can be contacted with the electrode system.
BACKGROUND OF THE INVENTION
Periodical monitoring of blood glucose levels is needed for the diagnosis and prophylaxis of diabetes mellitus. Adopting colorimetry or electrochemistry as their operation principle, strip type analyzers are conventionally used to determine glucose levels in blood.
Such a calorimetric principle is based on the glucose oxidase-colorimetric reaction represented by the following reaction formula 1:
Glucose+O
2
→Gluconic acid+H
2
O
2
(Glucose Oxidase Catalysis)
H
2
O
2
+Oxygen Receptor→(oxidized) Oxygen Receptor+2H
2
O (Peroxidase Catalysis)  Reaction Formula 1
In the presence of oxygen, as illustrated in the above formulas, glucose is oxidized with the aid of glucose oxidase to produce gluconic acid and hydrogen peroxide. From the hydrogen peroxide, oxygen molecules are transferred to an oxygen receptor (chromophore) by the catalysis of peroxidase. As a result of the oxidation, the chromophore changes color, and its color intensity is the basis of the quantitative analysis of blood glucose levels.
In order to utilize this calorimetric principle, however, precise care must be taken as to sample transport, pre-treatment, quantity, reaction time, and coloration starting time. In addition, blood coagulation or various interfering materials, including uric acid, ascorbic acid and bilirubin, may disturb the calorimetric analysis. Furthermore, the accompanying photometry has the fundamental limitation that its analytical accuracy and precision is lowered at high and low concentrations of samples. With these problems, the calorimetric analysis is known to be inappropriate for accurate quantification.
To avoid the problems that the calorimetric analysis has, electroanalytical methods were chemistry was introduced to biosensors. Over the calorimetric biosensors, the electrochemical biosensors have the advantage of being higher in selectivity and sensitivity, being able to measure the colored or turbid samples, without pre-treatment, and being able to perform accurate analysis within a short period of time. In order to better understand the background of the invention, the electroanalytical chemistry on which the second-generation biosensor will be described in conjunction with the following formula 2 and FIG.
1
. In contrast to the first-generation biosensor which uses oxygen as an electron transfer mediator, the second-generation biosensor takes advantage of an electron transfer mediator selected from the group comprising ferrocene, ferrocene derivatives, quinones, quinone derivatives, organic conducting salts, and viologen.
 Glucose+GO
X
-FAD→Gluconic acid+GO
X
-FADH
2
GO
X
-FADH
2
+Electron Transfer Mediator (Oxidized)→GO
X
-FAD+Electron Transfer Mediator (Reduced)  Reaction Formula 2
In Reaction Formula 2, GO
X
represents glucose oxidase, and GO
X
-FAD and GO
X
-FADH
2
are the oxidized and reduced forms of the glucose oxidase, respectively, because FAD (flavin adenine dinucleotide) is the active site of glucose oxidase. In
FIG. 6
, there is an electron transfer system for the electrochemical analysis of blood glucose levels, in which glucose oxidase and ruthenium are used as electron carriers from glucose to an electrode. Glucose is oxidized to gluconic acid by the catalytic action of glucose oxidase while the active site, FAD, of the glucose oxidase is reduced to FADH
2
, which transfers its electron, in turn, to the electron transfer mediator while being returned to the oxidized form FAD. The reduced electron transfer mediator ruthenium is diffused to the surface of an electrode. At the surface of the electrode, measured is the current generated when the oxidation potential of the reduced electron transfer mediator is applied. The oxidation potential is in the range of −0.2 to 0.2 V versus a reference electrode, so that the influence of ascorbic acid and uric acid, which have oxidation potentials higher than 0.3 V and 0.4 V, respectively, can be excluded.
In contrast to the first-generation biosensor, therefore, the second-generation biosensor is not affected by oxygen. In addition, the second-generation biosensor enables the non-selective catalysis of an oxidase and uses an electron transfer mediator, which has such appropriate redox potentials as to reduce the error caused by interfering materials, so that the measured potentials can be used to accurately determine the analytical quantity of interest. However, problems are also found in the second-generation biosensor. Because the electron transfer mediator, after the electron transfer to and from the oxidase, must diffuse to the surface of the electrode to detect the electrochemical change, a large quantity of the electron transfer mediator is needed. The abundance of the electron transfer mediator may alter the three-dimensional structure of the oxidase, resulting in a decrease in the activity of the enzyme. Another problem with most electron transfer mediators is high reactivity with electrode-activating materials in the blood.
Most of the glucose sensors, which are commercially available to date, follow the principle of the second-generation biosensors. Since the electron transfer mediators employed in most commercially available glucose sensors to be oxidized at potentials similar to oxidation potentials of the interfering materials within blood, such as ascorbic acid, acetaminophene and uric acid, the influence of the interfering materials within blood is not completely eliminated. To circumvent these problems, some commercially available glucose sensors take advantage of capillarity in introducing blood samples thereinto, but suffer from the disadvantage that their fabrication is complicated because there is required the process of coating hydrophilic polymers onto hydrophobic supports to achieve the introduction.
Recently, intense attention has been paid to the use of immunochromatographic methods in biosensors. In an immunochromatographic biosensor, a porous membrane is provided to form an electrode in its upper part and a sample pretreatment layer in its lower part. Without additional operations for pretreatment, the sample was moved through chromatographic action, during which a target material of the sample can be quantitatively determined by the change in electrical quantity of the target material (Cui, G., Kim, S. J., Choi, S. H., Nam, H., Cha, G. S, and Paeng, K. J.,
Anal. Chem.,
2000, 72, 1925-1929; Cha, G. S. et al., U.S. App. Ser. No. 09/381788 now U.S. Pat. No. 6,210,907; Farm, M. L., Rorad, O. H., Park, H., International Pat. WO 00/00827 2000). It is, however, difficult to introduce an electrode and a pretreatment layer together to a porous membrane for chromatographic motion. To relieve the difficulty, an electrode is formed on a plastic substrate while a porous membrane is used for a sample introducing part. This technique, however, is disadvantageous in that the electrode does not adhere well to the porous membrane of the sample introducing part that the sensor is poor in reproducibility. Additionally, it takes a long period of time for a sample to travel through the pretreatment layer t

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