Optical biosensor.

p0001The invention relates to an optical biosensor.

p0002The invention is based on the object to determine an analyte to provide a sensor that is versatile and in particular for measurements in flow chambers and directly in the solution and is suitable for determining glucose concentrations.

p0003This object is solved in an optical biosensor comprising an enzyme with an indicator bound to this, which reflects the change of an indicator quantity during a reaction catalyzed by the enzyme reaction in the analyte. Advantageous configurations of the optical biosensor of the invention are subject of the claims.

p0004In the biosensor according to the invention the enzyme is used to catalyze a reaction in the analyte. allows the detection of this reaction the antibody bound to the enzyme, ie the marking indicator which indicates a characteristic of the response indicator variable. Of course, the selection of the respective indicator is made such that its display provides a quantitative measure of a Analytbestandteils. For example, for the determination of glucose concentrations used as the enzyme glucose oxidase to which an indicator, a fluorescent dye is attached, showing a dependent of the oxygen concentration fluorescence. By the masses of the fluorescence intensity of the dye-labeled enzyme, a determination of the glucose concentration can be performed, as is consumed during the catalyzed by the enzyme reaction of this oxygen. So converts the formula used to determine glucose glucose glucose and oxygen into gluconolactone and hydrogen peroxide. With increasing glucose concentration in the analyte, the oxygen consumption is increased during this reaction, so that the fluorescence intensity is correspondingly greater. As regards the oxygen quenching of the fluorescence, so much oxygen in a low fluorescence, however little oxygen leads to a strong fluorescence. The biosensor according to the invention, it is possible to determine the concentration of a Analytbestandteils in a solution directly. For this purpose, the labeled enzyme need only be placed in a sample liquid. In the case of glucose oxidase labeled with fluorescence, the fluorescence is then measured before and after addition of an analyte, which thus contains glucose. If a 3 mM glucose solution was added, for example, for example, an increase in fluorescence of 60% are observed.

p0005Preferably, the labeled enzyme is immobilized on a support material in order to obtain a sensor surface. For optical purposes, therefore, the enzyme is applied according to an advantageous embodiment of the invention on a transparent support. In the transparent substrate may be a glass plate. Instead, however, other carriers may be used, for example, plastic films, or the selected enzyme is applied directly to the end of an optical fiber (single fiber or fiber bundle). Such a biosensor can be used directly for measurement in a flow-measurement. The mounting of the biosensor is made so that the labeled immobilized enzyme, eg glucose oxidase, the sample solution has, while the carrier is directed to a suitably provided Fluoreszenzmeßanordnung, a fluorometer back. The fluorescence of the labeled enzyme is for example excited by light having the wavelength of 405 nm through the transparent support and the fluorescence measured for example at 450 nm. It can Response times of about 50 to 100 seconds can be achieved. With immobilization of the enzyme, the maximum change in the fluorescence intensity decreases to about 20% (compared to 50% for direct measurement in solution). This is because the detected downstream of the Fluoreszenzmeßanordnung measurement signal has a higher interference signal component. This includes stray light, background fluorescence, so that the percentage change of the overall signal is smaller with a change of the concentration of the measured variable. Rules may range from about 0.01 to 2 mM be carried out in the case of glucose concentration. However, the sensor response is dependent here on the immobilization method used in each case.

p0006Preferably is used as a fluorescent indicator decacyclene. However, it can be any fluorescent dye may be used which shows a change of the fluorescence intensity with the concentration of oxygen.

p0007The enzyme layer is advantageously provided with a light-absorbing material, preferably a layer of activated carbon covered.

p0008According to the invention, the enzyme layer may be advantageously provided with a protective membrane, which is convenient by dipping into a solution. This verhinder undesirable reactions of the enzyme with the sample liquid. According to the thickness of the protective membrane, the sensor sensitivity or the analytical range of the sensor can be adjusted vorteilhalft. It is preferably a protective membrane made of cellulose acetate used (cellulose acetate in acetonitrile) or Cuprophan. However, other protective membrane materials are tight sets. The light-absorbing substance is preferably applied together with the protective membrane.

p0009As mentioned, the enzyme is in a preferred embodiment of the invention, glucose oxidase. Alternatively, the enzyme may be for example one of oxidases, oxygenases or oxidoreductases. This allows the determination of other Analyste in addition to the determination of glucose.

p0010As a fluorescent dye 3-COOH, -7-OH-coumarin is preferably provided after the carbodiimide method covalently to the enzyme (eg glucose oxidase) is bound. Further preferred 3-COOH-succinimidyl-7-OH-coumarin is used, which can be directly covalently bound to the enzyme. Here, each formed between the carboxy groups of the dye and the amino groups of the enzyme peptide bonds.

p0011As immobilization, the immobilization is preferably used with polyvinyl alcohol, wherein the labeled enzyme, for example with 7-hydroxycoumarin-labeled glucose oxidase, is immobilized in a hydrogel of polyvinyl alcohol photovernetztem. However, it can also be applied to other immobilization techniques, such as a cross-linking with glutaraldehyde is possible. Thus, each immobilisation may be used, in which the enzyme is bound in a kind of hydrogel while retaining the enzyme activity. For example, introducing a labeled enzyme in a polyacrylamide gel is possible.

p0012An inventively provided preferred method for producing an optical biosensor comprising the steps of providing a solution of an enzyme is produced with an indicator bound to this, the solution is mixed with polyvinyl alcohol solution, the solution mixture is applied to a transparent support and drying the solution mixture, and is photocrosslinking. Such a biosensor can be used directly in a flow-measurement.

p0013There may be other indicator quantities with the biosensor according to the invention are detected. For example, it can be determined instead of measuring oxygen consumption as during a reaction as the change in pH. For this pH indicators have to be immobilized directly on the enzyme. In the case of pH determination, other measuring arrangements may be used as Fluoreszenzmeßanordnungen, for example, a reflection or Absorptionsmeßanordnung.

p0014The invention will be further described hereinafter with reference to preferred embodiments and the drawing. In the drawings:<ul><li>Fig. 1 is a graph showing the change of the relative fluorescence by adding glucose to a solution of the labeled enzyme</li><li>Fig. 2 is a diagram illustrating a calibration curve of the relative fluorescence with respect to the glucose concentration for a biosensor with immobilized enzyme, and </li><li>Fig. 3 shows the structure of a glucose biosensor with immobilized enzyme.</li></ul>

p0015The invention is described further hereinafter by way of examples with glucose biosensors. Of course, as mentioned be determined with the inventive biosensor other analyte components. In the embodiments is always used as an enzyme with 7-hydroxycoumarin-labeled glucose oxidase, and it is measured the fluorescence intensity.

p0016In the example of FIG. 1, a biosensor without a carrier is used, ie, the labeled enzyme is dissolved in the sample solution. In the diagram two curves A and B are shown, which illustrate the relative fluorescence as a function of the wavelength. The trace A represents the Fluoreszenspekstrum without the addition of glucose. The measurement curve B has been added after addition of 3 mM glucose solution. A comparison of curves A and B indicates that the relative fluorescence rose with the addition of glucose by 60%. This type of determination of glucose may be referred to as a kinetic method.

p0017The calibration curve shown in Fig. 2 shows the relative fluorescence as a function of glucose concentration for a biosensor with immobilized labeled enzyme on a support. In the embodiment illustrated, the labeled enzyme has been immobilized in a hydrogel of polyvinyl alcohol photovernetztem. As illustrated, the fluorescence intensity will change by 20%. This change is much lower than that shown in Fig. 1 illustrated embodiment. This is because the optical measurement signal also contains scattered light and background fluorescence, which reduce the percentage change in the total signal. The illustrated in Fig. 2 calibration curve is not linear. However, a determination of the glucose concentration in the range of about 0.01 to 2 mM is well possible.

p0018In Fig. 3 an example of the structure of a glucose biosensor is illustrated. The sensor comprises a support 2, which is in the illustrated embodiment is a glass plate. On the glass flakes a labeled enzyme in the range of 1 cm² is applied. To this end, a solution of 400 U / ml glucose oxidase labeled an aliquot of 50 U / ml was taken and the ratio of 1: 2 was mixed with a ready solution of polyvinyl alcohol. After application, the assembly has been dried, and photo cross-linked. The biosensor thus produced can be clamped directly in a flow-measurement. The arrows 6 illustrate the sample liquid with which the enzyme reacts catalytically. By means of the arrows 8, 10 is supplied through the carrier 2 light to excite the fluorescence and the returning measuring signal is illustrated. For the excitation light is used with a wavelength of 405 nm. The fluorescence is measured at 450 nm.