DE19545547C1 - Enzyme membranes for bio-sensors - Google Patents

Abstract

Prodn. of an enzyme membrane comprises reaction of an enzyme with a poly-aldehyde, which has been obtd. by activation of a precursor.

Description

The present invention relates to a process for the production of enzyme membranes branches.

Enzyme membranes are suitable in combination with electrochemical sensors Manufacture of biosensors used to quantify organic substances, ins especially in biological liquids. These biosensors can z. B. in medical diagnostics, intensive care medicine, the environment control and food inspection.

The basics for the development of biosensors were laid back in 1962 (Clark, L., 1962). For the production of those used in the biosensors Enzyme membranes have numerous regulations, all of which are immobilization of the enzymes in or on a membrane have in common. The individual manufacturers Development methods differ from each other in particular in how the Immobilization of the enzymes is effected.

For example, enclosing an enzyme solution in a multilayer arrangement (EP-A-0 079 502). However, this possibility is because of low stability of the system is not advantageous.

Also known is an immobilization process in which enzymes in hydrogels be included (DE-A-38 12 442). However, this procedure has the Disadvantage that the gel is still very much after immobilization of enzymes swellable, resulting in a relative instability of the system as well as relative thick layers result. A usable functional stability is in the mei not to implement in most cases.  

The inclusion of enzymes in adhesives is also known (DD-AP-1 50 760). For Making an enzyme-coated film will apply liquid glue to the top brought the surface of a film, allowed to dry, the corresponding water-soluble Che enzyme or enzyme mixture applied and dry with the adhesive calmly. The adhesive layer contains non-uniformly distributed enzymes active particles, which has the disadvantage that the membrane is differently active Areas.

The enzymes can also be immobilized in that an aqueous Rige polymer emulsion or dispersion of emulsified acrylate or disperse system, in which enzymes are dissolved, is applied to a membrane and is dried by air flow and optionally a second membrane is pressed on (DE-C-44 10 809). This method has the disadvantage that the fixation of the Enzyme can be an incrustation, depending on the way the poly is prepared mer suspension or emulsion. This may result in unstable conditions Membranes.

The object of the present invention is therefore to provide a process for the production of enzyme membranes that are easy to carry out and enzyme creates membranes that are stable thanks to a homogeneous distribution of the Enzyme particles and reliable quality of the resulting enzyme layers to draw.

This problem is solved by a method according to claim 1. Before partial configurations result from the subclaims.

For long-term stability of the enzyme membranes, the enzymes in or be firmly fixed on the membrane, especially when used soluble enzymes. According to the invention, the enzymes are fixed covalently through the formation of a spatial network. This is through the implementation of an enzyme, possibly another amine component and polyaldehydes educated.  

According to the invention, non-enzyme proteins should be used under the amine component and enzymes, which, from the point of view of the enzyme membrane function, are the react tion of the sensor must not be understood. Exemplary for a non-enzyme protein are enzymatically inactive albumins, globules ne and vegetable stock proteins, especially bovine serum albumin (BSA), Hu Mans serum albumin, ovalbumin and methyl albumin. There is also Possibility of a second enzyme as an amine component in addition to the active enzyme to be used, however, from the point of view of the enzyme membrane function Must not influence the reaction of the sensor.

For the production of the enzyme membranes come as to be immobilized (active) Enzymes all enzymes known to the person skilled in the art, their use in enzyme membranes is useful, especially oxidoreductases or hydrolases for example glucose oxidase, lactate oxidase, urease, cholesterol oxidase, polya minoxidase, uricase, glutamate oxidase, lysine oxidase, laccase, glucoamylase, Mutarotase, β-galactosidase, invertase, glucose dehydrogenase, oligosaccharide hydrogenase, tyrosinase, catalase, monoamine oxidase.

According to the invention, polyaldehydes include compounds with at least two Understand aldehyde functions. The poly Aldehydes arise from the implementation of the method according to the invention their predecessors (precursors). The conversion of the precursors into active polyaldehyde de occurs through the reaction of the precursors with any oxidation medium, for example by their reaction with a periodate-containing solution (e.g. sodium periodate solution), with iodosylbenzene diacetate, iodosylbenzene, Osmi oxide or Pd (CH₃COO⁻). The use of periodate in is very preferred a concentration of 0.001 M / l to 0.6 M / l. As a precursor to the Polyaldehy de organic compounds can be used which are functional Contain groups with their oxidation at least two Aldehyde groups arise. Examples include: vicinal diol, 2-hydroxy-carboxylic acids, 2-keto-carboxylic acids or their mixture nice. Ethylene glycol, diethylene glycol and pinacol are particularly preferred preferably 2,6-dihydroxypimelic acid (dihydroxy-pentanedicarboxylic acid). As  Precursors for the polyaldehydes can also contain saccharide components of the enzymes, Di- or polysaccharides, such as sucrose or dextran and polyvinyl alcohols, as they are also represented in the membrane.

Compared to the direct addition of a polyaldehyde (e.g. glutaraldehyde) the "detour" over the precursor has the advantage that the formation of the poly aldehyde groups without side reactions and the resulting polyaldehyde is correctly defined. In addition, polyaldehydes can be obtained via the precursor that are normally not available.

According to the invention, any membrane which is customary in the field of enzyme membranes bran are used, but polysaccharide or polyvinylal are preferred alcohol membranes. Dextran, cellulose and cellulose hydrate are very preferred. (Cellophane), starch or inulin films.

It has proven to be advantageous to 0.5-5 mmol aldehyde groups per mmol Crosslink amino groups protein. In practice, however Simplification has proven itself to be 0.5-5 mmol aldehyde groups per mg protein network.

The inventive method for the production of enzyme membranes perform in two different ways:

A) Defined activation of the precursor and subsequent reaction of the activated th precursor with enzyme amino groups

The precursor is preferably mixed with a defined amount of oxidizing agent aqueous sodium periodate solution, mixed and during a for the quanti tative course of the reaction necessary time, preferably 2 hours to 3 Days, most preferably 12 to 24 hours, incubated. The resulting polyal dehyde is immediately combined with an enzyme solution and this mixture becomes on the membrane, preferably a polysaccharide or polyvinyl alcohol film, applied.  

B) activation of the precursor with simultaneous reaction of the activated pre with enzyme amino groups

In this case the oxidizing agent, preferably periodate, becomes directly the enzyme solution solution optionally added with the addition of the polyaldehyde precursor. To reprodizier Obtainable results need a constant temperature and a certain The pre-incubation time of the mixture must be observed before application to the membrane will. In the case of a sufficiently long pre-incubation period (approx. 120 to 240 Minutes) and an elevated temperature (approx. 20 to 60 ° C) the whole Oxidizing agent for the activation of the polyaldehyde precursors in the solution consumed and the structure of the membrane surface is not changed. At a shorter pre-incubation period (approx. 0 to 120 minutes) and a low one Temperature (0 to 20 ° C) is not all oxidant for activation the polyaldehyde precursor is consumed and activation also occurs the membrane surface, which is a covalent bond of the enzyme layer to the Pad (membrane) causes.

The incubation period for the production of the polyaldehyde can also be omitted. In in this case the enzyme is mixed with a defined amount of oxidizing agent, preferably aqueous sodium periodate solution, mixed and the mixture immediately onto the membrane surface, preferably a polysaccharide or polyvinyl alcohol foil, applied. It comes through the action of the oxidizing agent in the precursors of poly present in the enzyme and the membrane film aldehydes for the formation of aldehyde groups which are present in the enzyme which amine groups form a spatial network and to fix the Guide enzyme on or in the membrane surface.

On the other hand, it is also possible to use a pure solution of the enzyme on a mem apply bran film and cover immediately with another film. After this An oxidizing agent is preferred to dry on one of the foils from the outside wise periodate solution, applied. It diffuses inward and it a covalent bond is formed as described above (Ver wetting) that connects the enzyme with both foils.  

If mentioned in the two variants A) and B) enzyme or enzyme solution , this is either the enzyme to be immobilized (active) alone or a Mixture of the enzyme to be immobilized with an amine component. The Ver Use of the amine component has the advantage that more amino groups Formation of the network are available and the enzyme layer is more homogeneous becomes.

The enzyme membrane produced according to the invention can either be directly in one Biosensor used or rehydrated and with a second mem can be covered. After drying again, it can then be used for Construction of the biosensor can be used.

The enzyme membranes produced by the process according to the invention are characterized by the following advantages:

• - Structure of the enzyme layer is homogeneous and easily reproducible;
• - When using dextran as a polyaldehyde precursor there is a rapid drying of the enzyme layer, which is very quick and is reproducibly rehydrated;
• - Stability at room temperature, which they do without further action gets ready for use over a longer period of time;
• - high activity;
• - easy manufacture;
• - excellent diffusion properties

The invention will now be further with reference to the following examples wrote:

Examples Example 1: Application of the enzyme layer on a film

A cellophane layer is thoroughly deionized by multiple incubations washed water. This washed film is applied using a roller a smooth plate covered with nitrocellulose varnish is applied and attached to the  Edges fixed with a frame. Then they are left in a vertical position dry. Then circles are ge on the foil using a template desired diameter drawn with etching ink. After drying out the enzyme reaction mixture with a fine stick in each circle moderately applied. Everything is left in the fridge for at least 10 hours dry. Then the film is removed and left for about two hours Leave room temperature. Afterwards the film with 8% glycerol solution washed and trimmed according to the circles drawn. After Drying can use the enzyme membranes in a biosensor will.

In the manufacture of membranes in which the enzyme layer is mutual should be applied, the enzyme membrane, according to the above regulation has been made, covered in glycerol solution with a second film, smooth rolled and cut open. After drying, they become double-sided Enzyme membranes used for a biosensor.

Example 2: Preparation of a glucose oxidase membrane a) Activation of the polyaldehyde precursor

260 µl of a 0.5 molar solution of 2,6-dihydroxypimelic acid (adjusted to pH 7.0 using NaOH) with 650 µl of one 0.5 molar solution of sodium periodate mixed and sealed until kept dark the next day.

b) preparation of the reaction mixture

In a glass bottle, 100 mg glucose oxidase in 1 ml 0.01 M phosphate buffer, pH 7.0 dissolved. This corresponds to a concentration of 62.8 mg Glucose oxidase / ml solution and the volume activity is 5700 U / ml. 1055 µl this solution is mixed with 598 µl bovine serum albumin solution (concentration: 40 mg protein / ml) mixed and diluted with 863 µl deionized water.

The reaction mixture is prepared by preparing 440 µl of the above Enzyme solution combined with 91 ul of the precursor activated under a)  will.

The enzyme membranes are applied by applying 2 ul reaction mixture on a cellophane sheet with marked circles of 5 mm in diameter, like it described in Example 1.

If only an immobilization of the enzyme and not its solid connection with the film is required, the activation of the precursor according to step a) is so carry out that instead of 650 µl only 520 µl periodate solution is used and 130 µl deionized water are added to equalize the volume will.

In this example, using 2,6-dihydroxypimelic acid as a precursor Amount of 2 mmol aldehyde groups to 1 mg of the total used Protein (including the BSA used) cross-linked. The conversion of the Forerunner is quantitative. Immobilization of the enzyme on the film and its further crosslinking through the activation of the polysaccharides is through the formation of 1/2 mmol aldehyde groups from the polysaccharide part of the gluco seoxidase molecule and from the polysaccharides of the cellophane film (based on 1 mg of total protein used).

Example 3: Preparation of a lactate oxidase membrane a) Activation of the precursor

150 μl of dextran solution (100 mg / ml) in 0.01 M Phosphate buffer (pH 7.0) with 460 µl 29 mmol sodium periodate solution (in 0.01 M Phosphate buffer) mixed and sealed at laboratory temperature up to stored dark the next day. Immediately before mixing this solution with the subsequent enzyme solution, another 86 µl Added sodium periodate solution.

b) preparation of the reaction mixture

1 mg lactate oxidase (specific activity 35.7 U / mg)  dissolved in 35 µl 0.01 M phosphate buffer (pH 7.0) and with 70 µl solution of the precursor treated under a) for the preparation of the reaction mixture mixed.

The enzyme membranes are applied by applying 1 µl reaction mixture on a cellophane sheet with marked circles of 3 mm in diameter as it is described in Example 1.

If only an immobilization of the enzyme and not its solid connection with the film is required, the activation of the precursor according to step a) is so perform that only 460 ul of periodate solution are used and Volume compensation 86 µl deionized water (instead of another 86 µl periodic solution solution) can be added.

In this example, dextran is used as a precursor, so that dextran is statistically activated by the cleavage in every 7th polysaccharide unit. The Binding of the enzyme layer to the film is achieved by the formation of 0.5 mmol Aldehyde groups from the sum of the polysaccharides (dextran, saccharide content of the enzyme, cellophane film) for each mg of the enzyme used reached.

Claims (9)

1. A process for producing an enzyme membrane, characterized by the reaction of an enzyme with a polyaldehyde, the polyaldehyde being formed by activation from a precursor. 2. The method according to claim 1, characterized in that the implementation of the enzyme with the polyaldehyde with the addition of an amine component he follows. 3. The method according to claim 1, characterized in that the activation by the reaction of the polyaldehyde precursor with an oxidizing agent he follows. 4. The method according to claim 1 or 3, characterized in that the pre run a vicinal diol, a 2-hydroxy-carboxylic acid, a 2-keto-carbon acid, a polysaccharide or polyvinyl alcohol. 5. The method according to any one of claims 1-4, characterized in that the precursor is 2,6-dihydroxypimelic acid or dextran. 6. The method according to claim 3, characterized in that the oxidation medium is a sodium periodate solution. 7. The method according to claim 1, characterized in that the enzyme Glucose oxidase, lactate oxidase, polyamine oxidase, cholesterol oxidase, Urease, uricase, glutamate oxidase, lysine oxidase, laccase, glucoamylase, Mutarotase, β-galactosidase, invertase, glucose dehydrogenase, oligosac charid hydrogenase, catalase, monoamine oxidase or tyrosinase. 8. The method according to claim 2, characterized in that the amine com component is a non-enzyme protein or an enzyme that from the point of view the enzyme membrane function does not affect the detection reaction.   9. The method according to claim 8, characterized in that the niece zym protein bovine serum albumin, ovalbumin or human serum albumin min is.