STABILISATION OF ALDEHYDES

Abstract: Long chain aldehydes for use as co-factors in bio-luminescent enzyme systems are stabilised by forming a solution in an organic liquid which is at least a partial solvent for an organic polymeric material, coating the solution on a surface of appropriate polymeric material and at least partially evaporating the solvent phase. The polymeric or plastics surface can be incorporated in a cuvette for use in enzymatic reactions.

Description: STABILISft ION OF ALDHHiaSS This invention relates to methods for the stabilization of aldehydes. r Another aspect of the invention relates to a bio-luminescent sensor. 5 Assay teciiniques have been developed using enzyme systems which produce bio-luminescence. Thus for example a system coπprising luciferase and flavine reductase plus possibly NADH and E_*I can be employed using as a co-factor a long chain aldehyde. However, it has been found these long chain aldehydes are unstable, particularly in aqueous systems but even generally. Thus if the necessary amounts of 10 long chain aldehyde and enzyme system are measured out and placed in a series of lumin neter vessels (cuvettes) then, for the very small amounts of aldehyde added, it is found there is a steady degradation in activity over 4 weeks so that by the end of a storage period the system has lost 15 all activity. The reasons for this instability are not altogether clear. They may partly be an effect of evaporation and partly an effect of a small amount of the aldehyde being exposed to the atmosphere and undergoing degradation. Also controlled addition of the very small amounts required is difficult. Excess addition can inhibit rather than 20 prαrote the reaction. Addition of the aldehyde in solution so as to give controllable amounts is also ineffective as the presence of solvent often inhibits the bio-luminescent reaction. It has now been fcund that aldeihydes can be stabilised onto a 25 surface of an organic soluble polymeric material by dissolving the aldehyde in a solvent for both the aldehyde and the polymeric material, coating the solution onto the surface of the polymeric material and allowing the solvent to evaporate. The result"is surprising in that it would have been expected that this technique would have merely created 30 a very thin film of aldehyde on the surface which would be more subject _, to instability. Application of a similar solution to an inert surface such as glass tended to inhibit the reaction. It would appear that the \ amount of aldehyde which was effective in the technique of the invention where there was ccmbination with the surface of the plastics material was 35 sufficient in the case of glass to inhibit the reaction. According to the invention therefore there is provided a method for forming a stabilised long chain (6-14 carbon) aldehyde as co-factor for a bio-luminescent enzyme system characterised in that a solution of the factor in an organic solvent which is also at least partially a solvent for a plastics material is applied to a surface of the plastics material. The aldehydes used in this invention are those long chain aldehydes eπplσyed as co-factors in luminescent enzyme systems. The will have from 6 to 14 carton atoms and usually would be the straight chain aldehydes particularly n-dodecaldehyde and n-decylaldehyde (decanal). The polymeric or plastics surface can be a strip of plastics material or can be the surface or particularly the inside surface of the relevant portion of a luminαneter su i as a cuvette used in enzymatic reactions particularly a cuvette used for measurement of bio- luminescence. Such a plastics material must be at leas slightly soluble in the relevant solvent and materials such as polystyrene which are both clear and slightly soluble in cannon solvants are very suitable. The solvent will be a solvent for the aldehyde and also at least sufficient of a solvent at normal temperatures for the surface of the plastics material so that on application of the solvent t.o the surface, there is a slight dissolution which will re-deposit polymer as the above solvent evaporates. Solvents which would be suitable would be lower liquid etones such as acetone, hydrocarbons, benzene etc. Thus the solvent system can be a known solvent for a plastics material for example cycldhexanone or can be a mixed system comprising a mixture of a ketone and an alcohol for example ethanol. The solvent should be such that it is not likely to leave any residue after evaporation which would interfere with the enzymatic reaction. Those skilled in the art are well aware of bio-luminescent systems employing enzymes in which aldehydes are co-factors and the invention is applicable to most of these systems in that the invention is only concerned with the stabilisation of the aldehyde and does not otherwise affect its relationship with the reaction system. As indicated the choice of solvent will depend on the known properties of solvents. Many solvents are known to be excessively irihibitary of enzymatic reactions and therefore would not be used in case a trace of the solvent remaining as a residue frcm the evaporation would inhibit the reaction. Clearly the solvent as indicated above must be at least partially a solvent for the plastics material but no difficulty will be found in selecting solvents bearing in mind the well known properties of most organic liquids in regard to most of the common plastics materials. The portion of aldehyde to solvent is not apparently critical. It is an aspect of the invention that it permits the addition to the system of amounts of aldehydes which, if the aldehyde were added directly, would be excessive and inhibitory of the reaction. Apparently incorporation of the aldehyde into the plastics material results in a controlled release of aldehyde only in amounts which will prαrote the reaction rather than inhibit it. However, obviously the amount of aldehyde added in the solution should not be in such excess that it will inhibit the reaction even with incorporation into the plastics material. The appropriate upper limit can readily be determined by simple testing. It is also an aspect of the invention that the incorporation of at least part of the solvent system into the surface of plastics material tends to overccme the possible inhibition effect arising from the solvent that may be left after evaporation. The strength of the solution therefore and the-amount added is not apparently critical but in any case can readily be deter ined by simple testing bearing in mind the known properties of the relevant components. In utilising the stabilised aldehyde the surface of the plastics material can be brought into contact with the assay system containing enzyme, other co-factors, substrate and usually aqueous dispersent. Hcwever in seme instances it has been found that it is sufficient merely to add the other components to a cuvette or other assay vessel and the presence of vapour from the stabilised aldehyde/plastics surface is sufficient to allow the reaction to continue. The interaction of the solvent with the plastics material appears to form an amorphous reservoir phase which retains the aldehyde and possibly also some of the solvent and allows release of these in a manner which does not inhibit the reaction but does promote it. The presence of a second solvent for example a lewer alcohol (1-6 carbon atoms) appears to improve stability by increasing the total liquid content in the final "reservoir" phase bonded to the plastics surface possibly through reduced evaporation. So long as a reservoir phase is created the concentration and mixture of solvents can, as indicated above, vary widely. It is a further advantage of the invention that in a preoonstructed system to which a test fluid is to be added, the spacing of an aldehyde present in a stabilised form on the plastics surface away from the balance of the system enhances stability possibly because of avoidance of contact with aqueous ccπponents which can cause degradation of the aldehyde. A particularly valuable aspect to the invention is that a solution of the aldehyde can be applied to a portion of the inside wall of the cuvette or other portion of an assay kit and, after evaporation of the solvent to leave the aldehyde deposited and incorporated into the wall of the cuvette or other portion of the assay kit, one could apply to the same portion the enzymatic system so concentrating the light production over a limited area making it easier to determine the extent of light produced and consequently πaking the bio-luminescence assay much more accurate. In another aspect of the invention the ccπponents necessary for the enzyme system for a bio-luminescent assay are applied to a portion of a wall of a container for use in a lumincmeter so as to provide a concentration of these cccπponants over the given portion. On addition to the aqueous system to be subjected to the assay the light produced will be concentrated at the surface and will thus not be subject to interference by the aqueous system. For instance where the ccmponents of the enzyme system are distributed through the liquid as in previous assays the colour of the system could seriously interfere with the accuracy of the test. The enzyme system can be inrrobilized into a gel material hich in turn can be layered onto a portion of a container already treated in accordance with the invention in connection with the stabilization of aldehydes. Alternatively a cuvette provided with a portion of the wall bearing stabilised aldehyde can be provided with an inner wall over the specified portion and between the two walls there can be inserted the enzymatic system. The inner wall can be porous or perforated so permitting entry of the material to be assayed into the enzymatic system when it is placed in the cuvette or other measuring vessel. The luminometer can then read through the wall of the vessel directly onto the the narrow strip involved. Alternatively the enzyme system could be applied to the wall of the cuvette or other vessel and an inner wall applied, the surface adjacent to the enzyme being a plastic surface bearing stabilized aldehyde in accordance with the invention. Thus the enzyme possibly in the form of a gelled enzyme system could be placed at the bottαn of a cuvette with long chain aldehyde adsorbed onto a tight fitting porous disc above the gel. In another possible construction an L-shaped plate would be placed in a cuvette with the horizontal liπto attached for example fcy gluing or adhesive to the bottom. A space would be provided between the upright limb and the inner wall of the cuvette the width of the cαipartment, if necessary, being maintained by spacers, for example spacer discs one at the tcp and one at the bottom. These spacers could be integral with the upright or separately attached. The upright of the limb would be porous or perforated the pores or perforations being relative to the area to be scanned by a diode detector of an lumincmeter. One could then establish the luminescent system by providing on the wall of the cuvette which is the outer wall of the ccπpartment the stabilized long-chain aldehyde in accordance with the invention. One could then insert the L-shaped plate and attach to the cuvette and insert into the cccrpartment the luminescent enzyme system. In such a system one would have a specific dehydrogenase for the analyte in question. This could be adsorbed onto a cellulose or the enzyme could be in a gelling material which does not inactivate the enzyme. This system could also be applied to other bio-assay systems involving measurement of light factors such as luminescence or colour change. Colour change strips have been proposed for various chemical systems but the placing of a colour strip in a liquid to be assayed is not convenient for systematic determination where the change in the strip is to be analysed by a reflectance densitαneter or a lumincmeter. Example A solution was prepared ccataining by weight 90% cyclchexanone, 9% ethanol, and 1% decanal (n-decylaldehyde). 10/Λ of the solution was applied to the wall of a polystyrene commercial cuvette in a thin layer over the internal surface of the cuvette. The inside of the cuvette was then subjected to air drying or 20 minutes. The cuvette was then sealed and left for two hours. Thereafter there was added to the cuvette a bio- luminescent assay system with standard reagents in an amount of 1ml. The luminescence produced was compared with that produced by adding to a cuvette containing 1ml of the same assay system a very minor amount of decanal found by experimental testing to be that which would give the optimum luminescence. The procedure given above for the polystyrene cuvette was repeated in relation to a glass cuvette. As compared to the untreated cuvette in which decanal was added directly the luminescence produced for the glass cuvette was 1% and for the plastics cuvette was 70%. In this connection it should be noted that the amount of decanal added by adding lQμl of the solution would in itself have normally been highly inhibitory of the reaction. Polystyrene cuvettes treated with the solution according to the above procedure and stored four weeks were found to retain activity in respect of the aldehyde which was substantially that of the original cuvette immediately following treatment and evaporation of the solvent. In cuvettes in which a very small amount of decanal was added to the cuvette in the absence of solvent and then held for. four weeks under similar conditions there was a substantial loss of activity in the decanal over the relevant period.

Claims: CLAIMS: 1. A method for forming a stabilised long chain (6-14 carbon) aldehyde as co-factor for a bio-luminescent enzyme system <_haracterised in that a solution of the factor in an organic solvent which is also at least partially a solvent for a plastics material is applied to a surface 5 of the plastics material and the solvent is substantially evaporated. 2. A method according to claim 1 wherein the aldehyde is a straight chain aldehyde. 3. A method according to claim 2 vherein the aldehyde is n- dodecaldehyde or n-decylaldehyde. 104. A method according to any one of claims 1 to 3 wherein the cuvette is polystyrene and the solvent at least a partial solvent of polystyrene. 5. A method according to any one of claims 1 to 4 in which the solvent ccπprises a mixture of solvent and other organic liquid not iself 15 a solvent for plastics material. 6. A method according to claims 4 and 5 in which the solvent is a ketone. 7. A method according to claims 5 and 6 in which the solvent comprises a mixture of a ketone and a 1-6 carbon alcohol. 208. A method according to any one of claims 1 to 7 in which the plastics surface is the inner wall of a bio-luminescence assay cuvette. 9. A bio-luminescent assay device having a clear wall portion the inner surface of which portion bears an aldehyde stabilised in accordance with airy one of claims 1 to 8. 2510. A bio-luminescent assay device acoording to claim 9 characterised in that irrmediately adjacent to said surface or applied thereto are the reπaining components for a system for bio-luminesence assay.

Inventors: CASS ANTHONY EDWARD GEORGE, MANUEL DAVID CAMERON, CASS, ANTHONY, EDWARD, GEORGE, MANUEL, DAVID, CAMERON
Applicants: GRAND METROPOLITAN BIOTECH, GRAND METROPOLITAN BIOTECHNOLOGY LIMITED
Application year: 1986
Main classification: C12Q1
IPC codes: C12M1, C12Q1, G01N21, G01N33