PRODUCTION OF IMMOBILISED BACTERIOPHAGE

Abstract

Bacteriophage is covalently attached to a substrate by (a) combining (i) substrate with (ii) bacteriophage, wherein prior to or during the combining (i) or (ii) or both (i) and (ii) are activated, and wherein (b) during the combining the bacteriophage is contained within a liquid droplet of average diameter 150 microns or less.

Claims

1. A method of covalently attaching a bacteriophage to a substrate, comprising: (a) combining (i) substrate with (ii) bacteriophage, wherein prior to or during the combining (i) or (ii) or both (i) and (ii) are activated, and wherein (b) during the combining the bacteriophage is contained within a liquid droplet.

2-3. (canceled)

4. A method according to claim 1, wherein the substrate comprises particles of average diameter 500 microns or less.

5. A method according to claim 1, wherein the substrate comprises particles of average diameter 200 microns or less.

6. A method according to claim 1, wherein the liquid is aqueous.

7. A method according to claim 6, wherein the liquid is water.

8. A method according to claim 1, wherein the liquid is non-aqueous.

9. A method according to claim 8, wherein the liquid is or comprises a compound that is a gas at atmospheric pressures and 20 oC. and wherein the method is carried out under conditions of temperature and pressure such that the compound forms liquid droplets.

10. A method according to claim 9 wherein the covalently attached bacteriophage and substrate product is subjected to modified conditions of temperature and/or pressure so that the compound evaporates, yielding dry product.

11. A method according to claim 1, for attachment of bacteriophage to a water-sensitive substrate.

12. A method according to claim 11, wherein the substrate comprises a superabsorbent polymer.

13. A method according to claim 1, comprising activating the substrate and combining activated substrate with the liquid droplet.

14. A method according to claim 1, comprising activating the droplet and combining the activated droplet with the substrate.

15. A method according to claim 1, comprising activating both substrate and droplet and combining them

16. (canceled)

17. A method according to claim 1, comprising combining droplets of mass median diameter 1-200 microns with particles of mass media diameter 1-200 microns.

18. A method according to claim 17, comprising combining droplets of diameter 100 microns or less with particles of diameter 100 microns or less.

19. A method according to claim 17, wherein the ratio of the respective diameter of droplets and particles is from 1:3-3:1.

20-24. (canceled)

25. Apparatus for covalently attaching bacteriophage to a substrate according to the method of claim 1, comprising: a) means to generate droplets containing bacteriophage; b) (i) a plasma generator for generating a plasma in combination with a droplet activating station to contact the droplets of (a) with the plasma, or (ii) a plasma generator for generating a plasma in combination with a substrate activating station to contact the substrate with the plasma; and c) a chamber in which substrate and bacteriophage can be combined at the same time as or after contact with the plasma so as to form a covalent bond between the bacteriophage and the substrate.

26. Apparatus according to claim 25, adapted to generate liquid droplets of a solvent or solution that is a gas at room temperature and 20 oC. containing bacteriophage.

27. Apparatus according to claim 25, wherein the plasma generator of b (ii) is adapted for generating a plasma to activate the substrate.

28. Apparatus according to claim 25, wherein the plasma generator of b (i) is adapted for generating a plasma to activate the droplets containing bacteriophage.

29.-55. (canceled)

Description

[0213] The invention is now described with reference to the accompanying drawings, in which:

[0214] FIG. 1 shows a schematic diagram of apparatus for use in the invention for generation of activated polymer particles;

[0215] FIG. 2 shows a schematic diagram of apparatus of the invention for production of immobilised bacteriophage; and

[0216] FIG. 3 shows a schematic diagram of further apparatus of the invention for production of immobilised bacteriophage.

EXAMPLE 1

[0217] Apparatus was designed for immobilisation of bacteriophage (and other molecules) onto the activated surface of particles and filaments for the manufacture of bulk product. The apparatus was designed to permit the corona activation of materials, particularly particles, and reaction with bacteriophages or other viruses and substances to take place very rapidly, and within the lifetime of the induced free radicals.

[0218] Referring to FIG. 1, this shows a basic electrospray system comprising a high voltage supply to produce a corona between the induction electrode and the liquid nozzle, producing in operation a stream of polymer particles activated and ready for covalent attachment to bacteriophage.

[0219] FIG. 2 shows how the particle activator of FIG. 1 is integrated with a second liquid intake having opposite polarity.

[0220] Via inlet A is introduced a bacteriophage suspension at a suitable concentration and sufficient electric field so that emerging droplets are negatively charged. In parallel, via inlet B is introduced a polymer solution to be discharged through an electro-spray nozzle to form positively charged droplets—in proximity to the bacteriophage containing droplets.

[0221] In operation, particle surfaces are activated by corona discharge upon emergence from the electro-spray nozzles and combination with oppositely charged particles/droplets and immobilisation (covalent attachment) occurs within the reaction chamber. Through the reaction chamber, flow of drying gas facilitates transport and collection of particles.

EXAMPLE 2

[0222] A second apparatus for immobilisation of bacteriophage onto particles was similarly designed to permit the corona activation of materials, particularly particles, and reaction with bacteriophages or other viruses and substances to take place very rapidly, and within the lifetime of the induced free radicals.

[0223] In the second apparatus, shown schematically in FIG. 3, particle production using an electro-spray system is combined with a secondary corona stage.

[0224] The secondary corona stage has the same polarity and is situated to take advantage of particle flow using inert gas. A second spray nozzle is employed for bacteriophage droplet production with an opposite polarity and introduction of the bacteriophage droplets into the mixing chamber. Mixing of the charged bacteriophage droplets with the opposite charged polymer droplets results in rapid contact and combination in less than a second, leading to covalent attachments being formed.

REFERENCES

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