GB2318413A

GB2318413A - Gas or liquid analysis with reagents disposed in sequence

Info

Publication number: GB2318413A
Application number: GB9721837A
Authority: GB
Inventors: Tony Ernest Edmonds
Original assignee: Loughborough University; Loughborough University Innovations Ltd
Current assignee: Loughborough University; Loughborough University Innovations Ltd
Priority date: 1996-10-16
Filing date: 1997-10-16
Publication date: 1998-04-22
Anticipated expiration: 2017-10-16
Also published as: GB2318413A8; GB9721837D0; GB2318413B; GB9621562D0

Abstract

Gas or liquid samples 30 are analysed using a vessel e.g. a flask or capillary 10 containing reagents 21 - 23 disposed in sequence characterised by each reagent diffusing/reacting in total with the sample before the next reagent in the sequence is contacted. Unwanted reactions between neighbouring reagents are thus alleviated. The reagents may be arranged in distinct zones separated by neutral zones 44, 45.

Description

2318413 Sequential Chemical Reactions The present invention concerns

methods and apparatus for performing sequential chemical reactions, particularly, but by no means exclusively, for performing a simple one-step analysis of a sample.

Dry chemistry systems (e.g. Kodak Ektachern (RTM) and Ames Seralyser (RTn (Figure 1) are used to carry out simple one-step tests in clinical or other analyses. In such systems, the liquid sample is added to a multilayer 'pad' of dry reagents, the sample 10 is spread by a 'spreading layer' 20, is drawn into the reagent layers 3 1,3 2,3 3 by gravity and the capillarity/porosity of the medium which supports them. As the leading portion of the sample passes through each layer, the reagents are dissolved and a sequential series of chemical reactions occurs between the sample and the reagents, culminating in an unambiguous detectable indication of the target analyte when the sample reaches the detection layer or layers 40. However, as the sample encounters each new layer of reagent, part of the sample may still be encountering reagents in previous layers and it may be possible for unwanted chemical reactions to occur due to the mixing of reagents from different layers. Such approaches may be both costly and complex.

According to the present invention there is provided a method for performing a sequential set of -P-.comprising pi=inga sainple lo be analysed in a vessel containing reagents for performing an analysis, the sample moving relative to the reagents in order to contact each of them in sequence, the sample being in such a state as to allow a substantially complete diffusion of each reagent into the sample before the next reagent is incorporated.

The present inventor has found that by allowing the sample to retain its integrity (i.e. by retaining it in such a state as to allow a substantially complete diffusion of the reagent into the sample before the next reagent is incorporated), improved results may be achieved. As the sample moves forwards, it is allowed to interact with reagent zones rather than transiently pass through them, i.e. the reagent or a portion of it is incorporated into the sample volume, rather than the sample volume being incorporated into the reagent layers.

By allowing the sample to interact sequentially with the reagents and to maintain its integrity, the process of analysis is greatly improved. The person performing it requires little if any skill, and the manufacture of analysis kits is greatly simplified since complex multilayer pads having varying physical characteristics, e.g. porosity, need not be manufactured. In addition, since the sample maintains its integrity, the sample as a whole comes into contact with each reagent, allowing simple control of reagent concentrations, pH and reaction times.

Each reagent may react in sequence with the sample such that no unwanted chemical interactions occur between reagents. By ensuring that the entire sample is exposed to =eh zcagzuand Ibx each reaction is distinct, unwanted interactions betw=p different reagents may be prevented. 'Mis is difficult, if not impossible, to achieve by multilayer pad systems where individual parts of a sample come into contact with the reagents, the concentration of reagent and reaction time varying between each 'part' of a sample.

The method may be performed in solution, for example with a liquid sample and solid reagents, although of course it may for example be performed using a gaseous sample and solid reagents.

Each reagent may be fully dissolved before the next reagent in the series is dissolved.

Hence during an analysis of a sample with reagents A, B, C and D, as the sample incorporates each of the reagents in turn, whilst maintaining its integrity, any component of the system, including the analyte, may react first with A, then with B, C and D respectively to give a final result.

The reagents may be arranged in a series of distinct zones within the vessel.

At least two zones may be separated by at least one 'neutral' zone which has no chemical effect upon the sample.

At least one neutral zone may comprise a space between two reagentcontaining zones- At least one neutral zone may comprise a physical barrier between two reagent zones. The physical barrier may comprise a soluble matrix.

Reagent zones may be separated byneutral zoneswhich have no chemical effect upon the sample. Depending upon the nature of the apparatus used to perform the method, a neutral zone may for example comprise a space between two zones, or it may comprise a soluble matrix containing no reagent. Neutral zones may, for example, act solely to provide time to allow the previous reagent to complete its reaction with any analyte present.

The vessel may also comprise a sample container.

The vessel may be arranged such that the sample rises up the vessel, the first reagent being at a lower height than the final reagent.

In particular, three different methods and apparatus are provided. The first relies upon capillary action alone to bring the sample into contact with the reagent layers.

Internally-coated capiliM void method:

The vessel may comprise at least a capillary tube. The reagents may be bound to the inner surface of the capillary.

The reagents may be arranged as a series of zones along the inner surface of the capillary.

Capillary action occurs in fine-bore tubes or channels. The elevation of liquid in a capillary tube above the general level is given by the formula:

h 2 TeosO pgr wherein T is the surface tension, 0 is the angle of contact of the liquid with the capillary, p is the liquid density, g is the acceleration due to gravity, and r is the capillary radius. By liquid in this case is meant a liquid, mixture or solution.

Althought the prior art techniques use capillary action to help the sample migrate through the reagent layers, they work in conjunction with gravity whereas the present invention works against gravity. By altering the various factors which affect h, control of the rate at which the sample rises up a capillary and the height to which it rises may be effected. This control of the movement of a sample in a capillary allows for the fine control of sequential chemical reactions whilst maintaining the integrity of the sample. For example, in order to make a liquid rise flu-ther up a capillary, the surface tension may be increased, the angle of contact of the sample liquid may be made to approach 00, the density of the sample liquid may be decreased by precipitating out an insoluble product, or the capillary radius may be decreased. Thus the reagents themselves may alter the elevation of the sample, for example by changing the pH of a sample or precipitating out a substance. Thus the completion of a reaction involving one reagent may trigger an elevation of the sample and the incorporation of the next reagent.

Externally-coated MUL-cl vaid n=dw-&- ]be vessel may comprise a porous material. The porous material may be inert.

The porous material may be coated with the reagents. The porous material mey be coated on its outer surface with the reagents.

The reagents may be printed onto the outer surface of the porous matrial. The reagents may be printed onto the outer surface of the porous material by screenprinting.

The sample is drawn through the porous material by capillarity and dissolves out the reagents as it rises up the material. The incorporation of the reagents into the samples effects the requisite chemical reactions and brings about the detection of the analyte.

Neutral zones may be created by having areas of the porous material which are not coated with any reagent.

Immobilised Reagent Method:

The vessel may comprise a flask. The surface of the flask may bear the reagents. Alternatively or in addition, a support within the flask may bear the reagents.

The reagents may be arranged in a series of distinct zones within the vessel, thezones being mranged in a series of layers.

The reagents may be bound within a matrix. The matrix may be soluble. The matrix may be highly viscous or rigid.

]be reagents may be arranged as individual zones within the matrix such that they are dissolved sequentially by the solvent.

The reagents may be arranged in a series of distinct zones within the vessel, the.zones being an-amged in a series of layers bound within a soluble matrix, each layer being separated by a neutral zone having no chemical effect upon the sample, such that upon addition of a sample, each reagent is completely dissolved before the next reagent is dissolved.

The method may additionally compdse the step of determining the emission and/or absorption of electromagnetic radiation by the sample. Thus the state of the sample during and after the sequence of reactions may be assayed.

Also provided according to the present invention are apparatus for performing the method of the present invention.

The apparatus may comprise a vessel containing a set of reagents so arranged that a sample introduced into the vessel moves relative to the reagents and contacts and incorporates substantially the whole of each of them in sequence, the sample being in such a state as to allow a substantially complete diffusion of each reagent into the sample before the next reagent is incorporated.

The apparatus may additionally compdsing an emitter and/or detector of electromagnetic radiation. Thus for example a colorimetrie assay may be performed upon the sample, both during and after the sequence of chemical reactions and absorbance and emission of electromagnetic radiation (both direct and indirect) detected. The apparatus may additionally comprise waveguides for the electromagnetic radiation.

The apparatus may be used in a method according to the present invention.

Also provided is a kit of parts for apparatus according to the present invention.

The invention will be flather apparent from the following examples which show, by way of example only, three forms of method and apparatus for performing a sequential set of chemical reactions. Of the figures:

Figure 1 shows a prior art multilayer pad;

Figure 2 shows a internally coated capillary; Figure 3 shows an externally coated capillary; and Figure 4 shows immobilised reagents within a soluble matrux.

Int=ally cwted rinillary Ynidmrjhod The method comprises placing a sample solution 30 to be analysed in a capillary tube 10 having bound to its inner surface reagents 21,22,23 for performing an analysis, the sample moving relative to the reagents 21,22,23 in order to contact and incorporate substantially the whole of each one of them by fully dissolving them in sequence, the sample 30 being in such a state as to allow a substantially complete diffusion of each reagent 21,22 before the next reagent 22,23 is incorporated.

Each reagent 21,22,23 reacts in sequence with the sample such that no unwanted chemical interactions occur between reagents 21,22,23.

The reagents 21,22,23 are arranged in a series of distinct zones 41,42,43 within the capillary tube 10 separated by neutral zones 44,45 comprising spaces between reagent-containing zones 41,42 and 42,43, the neutral zones having no chemical effect upon sample 30.

Capillary tube 10 is arranged such that the sample 30 rises up it, the first reagent 21 being at a lower height than the final reagent 23.

Externally coated capiliM void method:

The method comprises placing a sample solution 30 to be analysed in a vessel 10 comprising inert porous material 50 bearing reagents 21,22,23 for performing an analysis, the sample moving relative to the reagent 21, 22,23 in order to contact and incorporate substantially the whole of each one of them in sequence, the sample 30 being in such a state as to allow a substantially complete diffusion of each reagent 21,22,23 before the next reagent 22,23 is incorporated.

The inert porous material is coated on its outer surface with the reagents 21,22,23 which have been printed onto the outer surface of the porous material by screen-printing.

Each reagent 21,22 is fully dissolved before the next reagent 22,23 in the series is dissolved.

The reagents 21,22,23 are arranged in a series of distinct zones 41,42,43 on the vessel 10, separated by neutral zones 44,45 comprise spaces between reagentcontaining zones 41,42,43 which have no chemical effect upon sample 30.

The vessel 10 also comprises a sample container 50.

The vessel 10 is arranged such that the sample 30 rises up the vessel 10, the first reagent 21 being at a lower height than the final reagent 23.

Immobilised Reagent Method The method comprises placing a sample solution 30 to be analysed in a flask 10 bearing on its surface bound within a soluble matrix reagents 21, 22,23 for performing an analysis, the sample moving relative to the reagents 21,22,23 in order to contact and incorporate suubstantially the whole of each one of them in sequence, the sample 30 being in such a state as to allow a substantially complete diffusion of each reagent 21,22,23 before the next reagent 22,23 is incorporated.

The reagents 21,22,23 are arranged in a series of distinct zones 41,42,43 within flask 10, physically separated by neutral zones 44,45 comprising a soluble matrix which has no chemical effect upon the sample 30.

The reagents 21,22,23 in their zones 41,42,43 are arranged in a series of layers 41,42,43,44,45 bound within a soluble matrix, each reagentcontaining layer 41,42,43 being separated by a neutral layer 44,45 having no chemical effect upon the sample 30, such that upon addition of a sample 30, each reagent 21,22 is completely dissolved before the next reagent 22,23 is dissolved.

Claims

1. A method for performing a sequential set of chemical reactions comprising placing a sample to be analysed in a vessel bearing reagents for performing an analysis, the sample moving relative to the reagents in order to contact and incorporate substantially the whole of each of them in sequence, the sample being in such a state as to allow a substantially complete diffusion of each reagent into the sample before the next reagent is incorporated.

2. A method according to claim 1 wherein each reagent reacts in sequence with the sample such that no unwanted chemical interactions occur between reagents.

3. A method according to any one of the preceding claims wherein it is performed in solution.

4. A method according to claim 3 wherein each reagent is fully dissolved before the next reagent in the series is dissolved.

5. A method according to any one of claims 1-4 wherein the reagents are arranged in a series of distinct zones within the vessel.

6. A method according to claim 5 wherein at least two zones are separated by at least one neutral zone which has no chemical effect upon the sample.

7. A method according to claim 6 wherein at least one neutral zone comprises a space between two reagent-containing zones.

8. A method according to either one of claims 6 or 7 wherein at least one neutral zone comprises a physical barrier between two reagent zones.

9. A method according to claim 8 wherein the physical barrier comprises a soluble matrix.

10. A method according to any one of the preceding claims wherein the vessel also comprises a sample container.

11. A method according to any one of the preceding claims wherein the vessel is arranged such that the sample rises up the vessel, the first reagent being at a lower height than the fmal reagent.

12. A method according to any one of the preceding claims wherein the vessel comprises a capillary tube.

13. A method according to claim 12 wherein the reagents are bound to the inner surface of the capillary.

14. A method according to any one of claims 1 - 11 wherein the vessel comprises a porous material.

15. A method according to claim 14 wherein the porous material is inert.

16. A method according to either one of claims 14 or 15 wherein the porous material is coated with the reagents.

17. A method according to claim 16 wherein the porous material is coated on its outer surface with the reagents.

18. A method according to claim 17 wherein the reagents are printed onto the outer surface of the porous material.

19. A method according to claim 18 wherein the reagents are printed onto the outer surface of the porous material by screen-printing.

20. A method according to any one of claims 1-11 wherein the vessel comprises a flask.

21. A method according to claim 20 wherein the surface of the flask bears the reagents.

22. A method according to either one of claims 20 or 21 wherein a support within the flask be= &c =gent.,

23. A method accoding to either one of claims 21 or 22 wherein the reagents are arranged in a series of distinct zones within the vessel, the zones being arranged in a series of layers.

24. A method according to any one of claims 20723 wherein the reagents are bound within a matrix.

25. A method according to claim 24 wherein the matrix is soluble.

26.. A method according to any one of claims 20-25 wherein the reagents are arranged as a series of layers bound within a soluble matrix, each reagent-containing layer being separated by a neutral layer having no chemical effect upon the sample, such that upon addition of a sample, each reagent is completely dissolved before the next reagent is dissolved.

27, A method according to any one of the preceding claims additionally comprising the step of determining the emission and/or absorption of electromagnetic radiation by the sample.

28. Apparats for performing the method of any one of the preceding claims.

29. Appamtus acwr lo úlaim 28 for performing a sequ=tul wl ú-.x-f chemical reactions comprising a vessel containing a set of reagents so arranged that a sample introduced into the vessel moves relative to the reagents and contacts and incorporates substantially the whole of each of them in sequence, the sample being in such a state as to allow a substantially complete difflision of each reagent into the sample before the next reagent is incorporated.

30. Apparatus according to either one of claims 28 or 29, additionally comprising an emitter and/or detector of electromagnetic radiation.

31. Apparatus according to any one of claims 28-30 used in a method according to any one of claims 1-27.

32. A kit of parts for apparatus according to any one of claims 28-3 1.