DE19647863A1 - Bio-sensor with immobilised enzyme, etc. - Google Patents

Abstract

A biosensor in which a biocomponent has been immobilised on a sensor surface by means of a transglutaminase using a spacer is claimed.

Description

The invention relates to bioassays on which biocompo with transglutaminase using Spacer molecules have been applied, a process for the production of bioassays and their use dung.

Stabilization and immobilization of enzymes on assays, especially sensors, has been done so far often through cross-linking with bifunctional Rea genes, optionally with nucleophiles (e.g. amino groups) and / or electrophilic groups (carbonyl groups) of the protein react. Best known Vertre ter is glutardialdehyde. This method has the after partly that it is completely unspecific, too bad  control and is usually a drastic act loss of activity of the immobilized enzymes leads.

The object of the present invention is therefore in applying biocomponents to assay surfaces easy way to immobilize, with none at all Loss of activity of the immobilized component com men should.

This task is solved by a bioassay according to Claim 1, d. H. by an assay on the bio components using transglutaminase using have been immobilized by spacer molecules. Continue this task is accomplished by a method according to patent claim 9 and the use of the biosensor according to Claim 16 solved. Advantageous design conditions result from the subclaims.

The inventors found that with the help of Transglutaminase biocomponents very gently immobi can be lized. It is preferred here if bacterial transglutaminase (MTGase) used becomes. Mammalian transglutaminase is also possible. The MTGase is a microbial Enzyme with a molecular weight of 40 kDa, a pI from 8.9 and a pH optimum from 6 to 7. The enzyme catalyzes the reaction of a γ-carboxamide group of Glutamine residues with ε-amino groups from lysine residues. This creates intra- and intermolecular isopeptide bonds. However, leave some biocomponents only under reducing conditions (addition of Polymerize dithiothreit). Instead of Lysinre primary aliphatic amino groups can also be used network. In the presence of primary aminogrup pen the γ-carboxamide group becomes the glutamate residue de  laminated. The purification of transglutaminase (EC 2.3.2.13) from microorganisms is described by "Ando et al., Agric. Biol. Chem., 53 (10), pp. 2613-2617 (1989) " described.

According to the invention, among those to be immobilized and polymers to be stabilized with biocomponents Amino functions (e.g. polypeptides, proteins, enzymes, Receptors, hapten-carrier protein conjugates, immunoglobulin bulins, antibodies), biogenic amines or amino functions nalized nucleic acids are understood. In before Zugze way it is enzymes or Enzymge mix, very preferably around maltose phosphorylase (MP), Mutarotase (MR) and glucose oxidase (GOD) and Gemi nice of it.

According to the present invention, under the The term bioassay means both biosensors and assays such as microtiter plates, test tubes or test sticks Understood. However, biosensors are preferred.

Under the term "(bio) sensor" or "(bio) sensor According to the invention, transducer surface chen (e.g. electrodes or optrodes), chips (wafers) or Piezocrystals (Microbalances) understood who the.

According to the invention, the "spacers" are (Synthetic) polypeptides with a share of gluta min or lysine, e.g. B. poly-L-glutamine or poly-L-Ly sin. It is preferably a Ge mix of different spacers, preferably poly-L-glutamine plus poly-L-lysine. A polymer can also be used as a spacer serve with aliphatic amino groups.  

To immobilize a biocomponent on a Sensor surface becomes a mixture comprising Biokom component, spacer, MTGase in solvent, preferred Buffer or water, dissolved on the sensor surface applied. It is also a sequential immobilizer possible. In this case, the Biocomponent applied and then with spacers and Crossglutaminase cross-linked.

The immobilization of the biocomponents on the upper surfaces, in particular the sensor surface, is carried out in a very preferred manner in that

• - The bio component or a mixture of bio components is brought into solution,
• - The spacer or the spacer mixture in one Buffer (e.g. citrate buffer) used in solution is not
• - Aliquots of both solutions are put together pipetted,
• - The transglutaminase dissolved in buffer is given and
• - The resulting solution on a sensor upper surface is applied.

However, it is also possible to use the spacer and / or not to first bring the transglutaminase into solution, but immediately move to the dissolved biocomponent ben. It is also possible to add the spacer in solution bring and the biocomponent and / or the transglu taminase, preferably to add the MTGase in solid form. It is also possible to use both spacers and bio Add solid component to the dissolved MTGase.

The sensor is preferably surface of the sensors around a platinum electrode. The  The quantity applied to the electrode is between 1 and 20 µl, preferably 1-10 µl. Then the elec tread in a container of high humidity ("Moist vessel ") incubated and stored to dry out the applied solution on the electrode other.

When adding the spacer, preferably poly-L-lysine and / - or poly-L-glutamine, to the biocomponent (preferably: Enzyme), are formed by the reaction of the polypepti de using transglutaminase networks with each other in which the biocomponents to be immobilized are included. These networks differ fundamentally different from cross-linked casein gels. Of the negative effect of the swelling of the layer in the Measurement solution, which too large diffusion paths and long response times and low signal intensity did not occur.

The required amount of spacer, amount of biocomponent and Amount of transglutaminase must be individual can be determined by preliminary tests.

In addition to the immobilization and stabilization of En The transglutaminase can be zymmen in a preferred manner used to make hapten-protein conjugates, anti body and biogenic amines on surfaces permanently and to immobilize it stably. Cross-linking too adsorptively immobilized protein layers (e.g. Se rumalbumin, casein) can be done, what analyti cal applications is important because unspecific Bindings and the signal caused thereby this way can be reduced.  

The advantage of the present invention is that with the help of transglutaminase, especially the microbial transglutaminase, biocomponents can be immobilized very gently. Through the The choice of reaction conditions can be cross-linked be controlled very precisely. The use of the spa cer leads to the formation of networks in which the biocomponents while maintaining their full Functionality can be immobilized. The immobilization covalent connection takes place before moves over lysine and glutamine residues, or through a fold inclusion. Through the formation of networks it is even possible to use proteins without or only with few glutamine or lysine residues on the surface to immobilize. It is also advantageous that the bioassays produced have a high storage stability to have. The use of the spacer also brings the Advantage that it does not have sufficient substrate components ten, e.g. B. on the surface of enzymes, replaced and at the same time a distance between the biocomponents ten effects. For example, there is a distance between Enzymes are an advantage because the enzymes loosen up Composite can move better, what with rigid, di right networking is no longer possible and to acti loss of viability.

The invention will now continue with reference to the Figu described, which show:

Fig. 1 Stability of phosphate layer sensors comprising a crosslinked with glutaraldehyde enzyme; individual provisions are shown

Fig. 2 Stability of phosphate sensors; Immobilization of the enzyme layer using poly-L-glutamine, poly-L-lysine and MTGase; mean values and standard deviations from triplicate determinations are shown

Fig. 3 receiving a phosphate calibration curve; mean values and standard deviations from triplicate determinations are shown

Fig. 4 signal strength of a glucose sensor depending on the immobilized amount of GOD; the measured values shown are individual determinations from calibrations of the respective enzyme-coated electrode

Fig. 5 signal strength of a glucose sensor depending on the amount of poly-L-lysine / poly-L-glutamine; mean values and standard deviations from duplicate determinations are shown
The invention will now be further described with reference to the following examples.

EXAMPLE 1 Immobilization using glutardialde hyd (comparative example)

The enzymes maltose-phosphorylase, mutarotase and glu cose oxidase were used to make a phosphate sensors by cross-linking with glutardialdehyde on a platinum electrode using conventional metho the applied (e.g. Novel Enzyme Sensor for the determination of inorganic phosphate; N. Conrath et al., Anal. Chim. Acta 309, 1995, pp. 47-52). The location tion of the electrode was carried out at 4 ° C in the "moist container".  

0.1 mM phosphate and 0.2 mM malate in citrate buffer (50 mM, 0.1 M KCl, pH 6.5) served as the measurement solution. Measurements were made between 1 and 30 days after the electrode was manufactured. The result of the measurements is shown in FIG. 1 as a function of the age of the electrode. It can clearly be seen that the signal intensity of such enzyme electrodes decreases drastically with increasing storage time.

EXAMPLE 2 Immobilization using MTGase under Use of spacers

The enzymes maltose-phosphorylase (Mp), mutarotase (MR) and glucose oxidase (GOD) were used to produce a phosphate sensor by cross-linking with MTGase using poly-L-lysine (PLL) and poly-L-glutamine (PLG) Spacers applied to a platinum electrode. The chosen approach was: 64 units GOD, 53 units MR, 5.4 units MP, 0.075 mg PLL, 0.128 mg PLG, 0.12 units MTGase in 6 µl citrate buffer (pH 6.5). The solution was applied to a platinum electrode and incubated for 16 hours at 4 ° C. in a wet flask. The further storage of the electrode was also carried out at 4 ° C in a wet container. 0.1 mM phosphate and 0.2 mM maltose in citrate buffer (50 mM, 0.1 M KCl, pH 6.5) were used as the measurement solution. Measurements were made between 1 and 18 days after the electrode was manufactured. The result of the measurements is shown in FIG. 2 as a function of the age of the electrode.

In addition, a phosphate calibration curve was recorded with the phosphate sensor prepared above. 0.8 mM maltose in citrate buffer (50 mM, 0.1 M KCl, pH 6.5) and the corresponding amount of phosphate (0 to 0.100 mmol / l) were added. The result of the measurements is shown in FIG. 3.

From FIGS. 2 and 3 it can be seen that the Si gnalintensität a phosphate sensor according to the invention is indeed lower than that of a conventional electrode (see. Example l), but it has also been observed on prolonged storage, no significant loss of activity.

EXAMPLE 3 Optimization of an enzyme PLG / PLL network works

4-80 units of glucose oxidase (GOD) were mixed with 0.4 mg PLG and 0.2 mg PLL. After adding 0.1 units of MTGase in 6 µl citrate buffer (pH 6.5), the solution (8 µl final volume) was applied to a platinum electrode and incubated for 16 hours in a wet flask at 4 ° C. The measurements were carried out with a glucose concentration of 1 mM. The result of the measurements is shown in FIG. 4. From this it can be seen that the signal intensity is best for a sensor on which approximately 20 units of GOD have been immobilized.

EXAMPLE 4 Signal strength depending on the Amount of cross-linking reagents (spa cerium)

16 units of glucose oxidase (GOD) with a PLG and PLL amount of 0.08 to 1.20 mg (ratio PLG: PLL = 2: 1) were added. After adding 0.1 units of MTGase in 6 µl citrate buffer (pH 6.5), the solution (8 µl final volume) was applied to a platinum electrode and incubated for 16 hours in a wet flask at 4 ° C. The measurements were carried out with a glucose concentration of 1 mM. The result of the measurements is shown in Fig. 5. From this it can be seen that the signal intensity is best for a sensor in which approximately 0.7 mg PLL + PLG have been used as the spacer.

Claims (16)

1. Bioassay, wherein a biocomponent by means of Transglutaminase using a spacer immobilized on a sensor surface is. 2. Bioassay according to claim 1, wherein the biocomponents te is selected from a polymer with amino function, biogenic amines or aminofunctional based nucleic acids. 3. Bioassay according to claim 2, wherein the polymer with Amino function a polypeptide, protein, enzyme, Receptor, hapten-carrier protein conjugate or Is antibody. 4. Bioassay according to claim 3, wherein the enzyme from is selected from glucose oxidase, mutarotase, mal tose phosphorylase or a mixture thereof. 5. Bioassay according to one of claims 1 to 4, characterized in that the transglutaminase is a bacterial transglutaminase. 6. Bioassay according to one of claims 1 to 5, wherein the spacer contains a (synthetic) polypeptide Lysine and / or glutamine residues or a polymer with aliphatic amino groups. 7. Bioassay according to claim 6, wherein the spacer poly- L-lysine and / or poly-L-glutamine. 8. Bioassay according to one of claims 1 to 6, wherein the assay is a biosensor and the sensor upper  surfaces are selected from transducer surface chen (e.g. electrodes or optrodes), chips (Wa fer) or piezo crystals (microbalances) or a microtiter plate, test tube or test rod. 9. Process for the preparation of a bioassay one of claims 1 to 8, wherein a mixture including biocomponent, spacer and transgluta minase in solution applied to a surface becomes. 10. The method according to claim 9, wherein as a biocompo nente a polymer with amino function, a bioge nes amine or amino functionalized nucleic acid be used. 11. The method of claim 10, wherein the polymer with amino function a polypeptide, protein, En zym, receptor, hapten-carrier protein conjugate, Is immunoglobulin or antibody. 12. The method of claim 11, wherein the enzyme from is selected from glucose oxidase, mutarotase, mal tose phosphorylase or a mixture thereof. 13. The method according to any one of claims 9 to 12, with a (synthetic) polypeptide as spacer with lysine and / or glutamine residues or a butt lymer used with aliphatic amino groups becomes. 14. The method of claim 13, wherein the spacer Is poly-L-lysine and / or poly-L-glutamine.   15. The method according to any one of claims 9 to 14, wherein the assay is a biosensor, the top of which surfaces are selected from transducer surface chen (e.g. electrodes or optrodes), chips (Wa fer) or piezo crystals (microbalances) or a microtiter plate, test tube or test rod. 16. Use of the biosensor according to one of the claims che 1 to 8 in biosensor technology.