JPS63500537A - Stabilization of aldehydes - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] The present invention relates to a method for stabilizing aldehydes.

Another aspect of the invention relates to bioluminescent sensors.

Analytical techniques using enzyme systems that produce bioluminescence have been developed. So, for example , by using long-chain aldehydes as cofactors, luciferase, flavi reductase, and in some cases a system consisting of NADH and FMN. Can be adopted. However, this long-chain aldehyde is especially useful in aqueous systems. It is unstable both in general and in general. Therefore, the required amount of long chain aldehyde and the enzyme system are weighed out into a series of luminometer vessels (cuvettes). For a very small amount of aldehyde added, there was a constant decrease in activity over 4 weeks. As a result, the system loses all activity at the end of the storage period. . The reason for this instability is not always clear; it is partly an effect of evaporation; Some are the result of exposure to small amounts of aldehydes or the atmosphere and their deteriorating effects. Also , it is difficult to control the very small amount of addition required, there is an excess of addition. This results in suppressing the reaction rather than promoting it.

Aldehydes can also be added into the solution to give a controllable amount , are inefficient because the presence of solvent often inhibits the bioluminescent reaction. .

Now, aldehyde is used as a solvent for both aldehyde and polymer material. The solution is applied to the surface of the polymeric material and the solvent is evaporated. Rudehydes can be stabilized on the surface of polymeric materials soluble in organic solvents. It has been found. This result suggests that this technique simply adds more unstable aluminum to the surface. It was thought that dehyde produced only a thin film of dehyde. When you think about it, it's amazing. When a similar solvent is applied to an inert surface like glass , tended to inhibit the reaction. There is a combination with plastic material surface An amount of aldehyde that is effective in the technique of the invention may inhibit the reaction in the case of glass. That was enough to stop it.

Therefore, according to the present invention, stabilized lengths as cofactors of bioluminescent enzyme systems A method for forming chain (6 to 14 carbon atoms) aldehydes in plastic materials. Add a solution of the above factors to an organic solvent, which is at least partially used as a solvent. A method is provided, characterized in that the method is characterized in that it is applied to a surface of a tick material.

The aldehyde used in the present invention is a long-term compound used as a cofactor in the 1-luciferase system. It is a chain aldehyde. This aldehyde has 6 to 14 carbon atoms and is usually a straight chain Aldehydes, especially n-dodecaldehyde and n-decylaldehyde (decanal ).

The polymeric or plastic surface may be a strip of plastic material and is Luminometers, especially cuvettes used for bioluminescence measurements. Such plastic material may be the surface, especially the inner surface, of the relevant part of the The material must be at least slightly soluble in the relevant solvent, be transparent and Materials such as polystyrene that are slightly soluble in common solvents are highly preferred. be. The solvent is a solvent for aldehydes and plastic materials at room temperature. The solvent must be at least sufficiently solvent for the surface of the When applied to the surface, there is a slight dissolution, and as the solvent evaporates, the polymer Reattach. Preferred solvents are lower liquid ketones such as acetone, hydrocarbons, benzene etc. Thus, solvent systems are known solvents for plastic materials, such as For example, it may be cyclohexanone, or a combination of a ketone and an alcohol, such as an alcohol. The solvent may be a mixed system consisting of a mixture with tanol, after evaporation, the enzymatic reaction It must not leave any residue that could interfere with the

Those skilled in the art will appreciate that aldehydes are cofactors. Regarding bioluminescent systems using enzymes, As is well known, the present invention relates only to the stabilization of aldehydes, and does not involve the reaction thereof. The present invention does not affect the relationship between the bioluminescent system and the bioluminescent system. It can be applied anywhere.

As mentioned above, the choice of solvent will depend on the known properties of the solvent. Many solvents are enzymes It is known to be overly inhibitory to the reaction and is therefore used as an evaporation residue. Obviously, the method shown above should not be used if traces of solvent remaining in the reaction inhibit the reaction. The solvent used must be at least partially solvent for the plastic material. However, the organic liquid surrounding most of the common plastic materials is Selection of a solvent is not difficult if intellectual properties are considered.

The ratio of aldehyde to solvent is clearly not critical. aldehyde Adds aldehyde to the system in an amount that would be excessive and inhibit the reaction if added directly. One of the features of the present invention is that it makes it possible to add Obviously, plus By incorporating aldehydes into the tic material, the reaction is promoted rather than inhibited. It becomes possible to control the amount of aldehyde that is released. however, The amount of aldehyde added in solution form is incorporated into the plastic material. It is clear that it must not be so excessive that it suppresses the reaction. appropriate upper limit can be easily determined by a simple test.

Evaporation by incorporating at least a portion of the solvent system onto the surface of the plastic material Another feature of the present invention is that it overcomes the inhibitory effect caused by the solvent left behind. There is one. Therefore, the potency of the solution and the amount added are clearly not critical, but In any case, a simple test can be carried out taking into account the known properties of the relevant components. can be easily determined by

When utilizing stabilized aldehydes, the surface of the plastic material is treated with enzymes, other Contact with the analytical system containing the agent, substrate and usually an aqueous dispersant. However, a If necessary, it is sufficient to add the other components to the cuvette or other The reaction continues even in the presence of stabilized aldehyde/steam from the plastic surface. I know that it can be done. Interaction between solvents and plastic materials Depending on the application, it may retain the aldehyde and possibly some of the solvent. , an amorphous reservoir phase is formed that releases these so as to accelerate rather than inhibit the reaction. It is thought that

The presence of a second solvent, such as a lower alcohol (1 to 6 carbon atoms), results in less evaporation. increasing the total liquid content within the final “reservoir” phase bonded to the plastic surface. It is thought that stability can be improved by

As long as a reservoir phase is created, the concentration and mixture of solvents can vary significantly as described above. can be obtained.

Yet another advantage of the present invention is that in the preconfiguration system to which the test fluid is added, the The aldehyde present in stabilized form on the surface of the stick is isolated from the rest of the system. This avoids contact with aqueous components that cause aldehyde deterioration. Another advantage is that it can promote stability.

A particularly important feature of the invention is that the solution of aldehyde is placed on the inner wall of the cuvette or It can be applied to other parts of the analysis kit and the aldehyde is placed in the cuvette after evaporation of the solvent. for attachment and incorporation into the walls of the test kit or other parts of the assay An enzyme system is applied to the above area to concentrate light generation in the area where the generated light is generated. Facilitating the measurement of light intensity and making bioluminescence analysis more accurate It is.

In another feature of the invention, the predetermined portion includes components necessary for an enzyme system for bioluminescence analysis. part of the container wall where these components are used in the luminometer, so as to provide a concentration section for the luminometer. When added to an aqueous system to be analyzed, the luminescence is concentrated on the surface and the aqueous system For example, components of enzyme systems can be analyzed in liquid form as in traditional analysis. , the color of the system can have an important bearing on the accuracy of the test.

The enzyme system is immobilized in a gel-like material, and the gel-like material is the main material for stabilizing aldehydes. It is layered over a portion of the container treated according to the invention. Alternatively, stabilized alkaline In a cuvette with a section of wall containing dehyde, an inner wall is placed over a specified section. It is possible to insert an enzyme system between the two walls. The inner wall is analyzed When the substance to be measured is placed in a cuvette or other measurement container, this substance enters the enzyme system. May be porous or perforated to allow entry. The luminometer may then be passed directly to the relevant strip through the wall of the container. be exposed.

Alternatively, the enzyme system can be applied to the walls of a cuvette or other container. Inside The wall, ie the surface adjacent to the enzyme, is coated with a polymer containing aldehydes stabilized according to the invention. It is a plastic surface. In this way, enzymes in the form of gelling enzyme systems can be can be placed at the bottom of the tray. At this time, the long-chain aldehyde is tightly placed on the gel. It is adsorbed to a porous disk. In other configurations, the L-shaped plate may be Place upright in a cuvette with a horizontal projection attached to the bottom by adhesive. A space is provided between the protrusion and the inner wall of the cuvette, if necessary.

The width of the compartment is determined by spacers, for example one at the top and one at the bottom. - held by a disk. This spacer may be integrated with the upright or attached to it. The upright part of the protrusion may be porous or have holes, and the holes and perforations The hole corresponds to the area scanned by the diode detector of the luminometer. One The wall of the cuvette, which is the outer wall of the compartment, is stabilized by the present invention. A light-emitting system can be created by providing a long-chain aldehyde. Next, L - Insert the shape plate into the cuvette and insert the luminescent enzyme system into the compartment. Ru. In such systems, the analyte of interest is Can contain dehydrogenase. This may be adsorbed onto cellulose or Alternatively, the enzyme may be placed in a gelling material that does not inactivate the enzyme.

This system is useful for other bioanalytical assays, including the measurement of optical elements such as luminescence or color change. It can also be applied to systems. Color-changing strips have been used in various chemical systems. One method that has been proposed for systems is the introduction of colored strips into the liquid to be analyzed. is analyzed by a strip reflectance densitometer or luminometer. It is inconvenient for systematic measurements.

Support 1 90% by weight cyclohexanone, 9% by weight ethanol and 1% by weight decana A solution containing alcohol (n-decylaldehyde) was prepared. Pour the solution into a 101Lfi poly Apply to the wall of a commercially available styrene cuvette.

A thin layer was formed on the inner surface of the cuvette. Next, empty the inside of the cuvette for 20 minutes. It was air-dried. The cuvette was then sealed and left for 2 hours. Then the cuvee Then, a bioluminescence analysis system containing standard reagents was activated to 1 mm. The generated light emission and 1 Experimental tests have shown that cuvettes containing mfL's analytical system have been tested to provide optimal luminescence. by adding a very small amount of decanal, which has been found to be The comparison was made with the same luminescence. The above procedure for polystyrene cuvettes can be repeated for glass cuvettes. Repeated for Cuvette.

Glass cuvettes compared to untreated cuvettes with direct addition of decanal. The luminescence produced was 1% for the plastic cuvette and 70% for the plastic cuvette. Met. What you need to pay attention to here is to add 10μ of the solution. The amount of decanal added by That's what I mean.

Polystyrene cuvettes treated with the solution as described above and stored for 4 weeks were For aldehydes, the activity of the raw cuvette immediately after processing and solvent evaporation is It was found that the same activity was maintained.

A very small amount of decanal was added to the cuvette without solvent and kept under similar conditions for 4 weeks. The cuvettes show a substantial decrease in activity in decanal over this period. It was below.

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Claims (10)

[Claims] 1. Stabilized long-chain (6-14 carbon atoms) alkaline compounds as cofactors in bioluminescent enzyme systems A method of forming dehyde, which also applies at least partially to plastic materials. A solution of the above factors dissolved in an organic solvent is added to the surface of the above plastic material. stabilized long chains, characterized in that they are attached to a surface and substantially evaporate the solution; How aldehydes are formed. 2. The method according to claim 1, wherein the aldehyde is a long chain aldehyde. . 3. The above aldehyde is n-dodecaldehyde or n-decyl aldehyde. The method according to claim 2. 4. the cuvette is polystyrene, and the solvent comprises at least one portion of the polystyrene. A person according to any one of claims 1 to 3, which is a partial solvent. Law. 5. The above solvents are solvents and others that are not themselves solvents for plastic materials. Claims 1 to 4 consisting of a mixture with an organic liquid of How to put it on. 6. 6. A method according to claims 4 and 5, wherein the solvent is a ketone. 7. A patent claim in which the solvent is a mixture of a ketone and an alcohol having 1 to 6 carbon atoms. The method according to scope items 5 and 6. 8. A patent claim in which the surface of the plastic is the inner wall of a bioluminescence analysis cuvette. The method according to any one of items 1 to 7. 9. It has a transparent wall portion, and the inner surface of the wall portion meets the requirements of claims 1 to 8. A bioluminescence analyzer carrying an aldehyde stabilized by either. 10. The components of the system value for bioluminescence analysis are directly adjacent to the above surface or the above table. The bioluminescence analyzer according to claim 9, which is attached to a surface.