Method and apparatus for generation of microparticles containing immobilized enzyme

Abstract

A method and an apparatus are described for the generation of microparticles containing an immobilized functional component, where the following measures are proposed: spraying a liquid (32) containing a soluble alginate and a functional component consisting of molecules or nanoparticles to generate a stream (60) of droplets, directing the stream (60) of droplets onto a precipitation bath (16) and capturing the droplets therein by application of high voltage (14), precipitating the droplets in the precipitation bath (16) via a precipitation liquid (18) containing an alginate complexing agent, such that the droplets are solidified to form microparticles (10) containing the functional component and extracting the microparticles (10) from the precipitation bath (16).

Claims

1. A method for generation of microparticles containing an immobilized functional component, the method comprising the steps of a) spraying a liquid which contains a soluble alginate and a functional component consisting of molecules or nanoparticles to generate a stream of droplets, wherein spraying the liquid comprises applying a gas stream to the liquid, thereby atomizing the liquid in the gas stream, wherein said spraying the liquid comprises providing the liquid as a static volume in a reservoir, applying the gas stream to the liquid in the reservoir, and adjusting size of the droplets by adjusting a property of the gas stream, b) directing the stream of the previously generated droplets through an electrically conductive nozzle which is connected as a counter electrode to a high voltage, wherein the high voltage is in a range of 3 to 80 kV, c) directing the stream of the droplets onto a precipitation bath and capturing the droplets therein by application of the high voltage, wherein a target electrode connected to the high voltage is positioned within the precipitation bath, d) precipitating the droplets in the precipitation bath by means of a precipitation liquid containing an alginate complexing agent, such that the droplets are solidified to form microparticles containing the functional component, and e) extracting the microparticles from the precipitation bath.

2. The method of claim 1, wherein the molecules forming the functional component are selected from the group of enzymes, coenzymes, mediators, stabilizers and dyes, and/or wherein the nanoparticles forming the functional component are sized in at least one dimension below 1 μm and/or are selected from the group of metal, metal alloy, metal oxide and carbon.

3. The method of claim 2, wherein the molecules forming the functional component are enzymes.

4. The method according to claim 1, further comprising providing a moving surface in the precipitation bath, wherein the moving surface is continuously loaded with a film of the precipitation liquid and the droplets are disposed onto the moving surface.

5. The method according to claim 1, wherein the target electrode is in the form of a submerged rotating drum.

6. The method according to claim 1, wherein the microparticles are formed with a dimension of less than 50 μm.

7. The method according to claim 6, wherein the microparticles are formed with a dimension in the range from 1 to 20 μm.

8. The method according to claim 1, further comprising separating microparticles of different size by an ion exchange process using different ions in the alginate complexing agent, wherein the ions are barium ions and calcium ions.

9. The method according to claim 1, further comprising providing the microparticles with a stabilizing shell made of a polymer material.

10. The method according to claim 1, further comprising discharging a suspension of the microparticles through an outlet of the precipitation bath.

11. The method according to claim 1, further comprising cleaning of the microparticles in a cleaning bath by means of an ion exchange process.

12. The method according to claim 1, further comprising depositing the microparticles in a layer of a diagnostic test element, wherein the layer is selected from a reagent layer or an electrode layer and the diagnostic test element is a glucose test element.

13. The method of claim 1, wherein the property of the gas stream includes at least one of velocity, temperature, and humidity of the gas stream.

14. A method for generation of microparticles containing an immobilized functional component, the method comprising the steps of: a) spraying a liquid which contains a soluble alginate and a functional component consisting of molecules or nanoparticles to generate a stream of droplets, wherein spraying the liquid comprises applying a gas stream to the liquid, thereby atomizing the liquid in the gas stream, wherein said spraying includes adjusting size of the droplets by adjusting at least one of velocity, temperature, and humidity of the gas stream, b) directing the stream of droplets onto a precipitation bath and capturing the droplets therein by application of high voltage, wherein an electrically conductive nozzle is connected as a counter electrode to the high voltage, wherein said directing the stream includes electrostatically charging the droplets by directing the stream of the droplets through the nozzle, wherein the droplets are generated before being electrostatically charged by the nozzle, wherein the high voltage to electrostatically charge the previously generated droplets with the nozzle is in a range of 3 to 80 kV, and wherein a target electrode connected to the high voltage is positioned within the precipitation bath, c) precipitating the droplets in the precipitation bath by means of a precipitation liquid containing an alginate complexing agent, such that the droplets are solidified to form microparticles containing the functional component, and d) extracting the microparticles from the precipitation bath.

15. The method of claim 14, wherein the molecules forming the functional component are selected from the group of enzymes, coenzymes, mediators, stabilizers and dyes, and/or wherein the nanoparticles forming the functional component are sized in at least one dimension below 1 μm and/or are selected from the group of metal, metal alloy, metal oxide and carbon.

16. The method of claim 15, wherein the molecules forming the functional component are enzymes.

17. The method according to claim 14, further comprising providing a moving surface in the precipitation bath, wherein the moving surface is continuously loaded with a film of the precipitation liquid and the droplets are disposed onto the moving surface.

18. The method according to claim 14, wherein the target electrode is in the form of a submerged rotating drum.

19. The method according to claim 14, wherein the microparticles are formed with a dimension of less than 50 μm.

20. The method according to claim 19, wherein the microparticles are formed with a dimension in the range from 1 to 20 μm.

21. The method according to claim 14, further comprising separating microparticles of different size by an ion exchange process using different ions in the alginate complexing agent, wherein the ions are barium ions and calcium ions.

22. The method according to claim 14, further comprising providing the microparticles with a stabilizing shell made of a polymer material.

23. The method according to claim 14, further comprising discharging a suspension of the microparticles through an outlet of the precipitation bath.

24. The method according to claim 14, further comprising cleaning of the microparticles in a cleaning bath by means of an ion exchange process.

25. The method according to claim 14, further comprising depositing the microparticles in a layer of a diagnostic test element, wherein the layer is selected from a reagent layer or an electrode layer and the diagnostic test element is a glucose test element.

Description

(1) The invention is further elucidated in the following on the basis of an embodiment example shown schematically in the drawings, where

(2) FIG. 1 is a schematic view of an apparatus for generation of microparticles;

(3) FIG. 2 is sectional view of a layered diagnostic test element comprising microparticles.

(4) FIG. 1 illustrates a system or apparatus for generation of microparticles 10 comprising a spraying unit 12, a high voltage unit 14, a precipitation bath 16 including a precipitation liquid 18 and a rotatable drum 20, and extraction means 22 for extracting precipitated microparticles 10 from the bath 16.

(5) The spraying unit 12, which can be positioned over the precipitation bath 16 by means of a stand 24, is provided with a gas inlet 26, an atomizer chamber 28 including a reservoir 30 for a feed liquid 32 and an aerosol outlet 34 ending in a discharge nozzle 36. The feed liquid 32 contains an aqueous solution of a soluble alginate, e.g. sodium alginate and at least one of enzyme selected from oxi-reductase-enzymes, dehydrogenase-enzymes or oxidase-enzymes and other functional molecules. Therewith, it is finally aimed to form beads of alginate complexes containing immobilized enzymes as (spherical) microparticles with a size or dimension (diameter) in the range from 1 to 20 μm.

(6) The high voltage unit 14 has a source 38 for a d.c. voltage in the range of 3 to 80 kV, preferred 10 to 60 kV which is supplied between a first port 38 and a second port 40. First port 38, suitably grounded, is connected to the drum 20 which thereby forms a target electrode 42, whereas second port 40 is connected to the nozzle 36 forming a counter electrode 44. Optionally, the first port 38 may be connected to the precipitation liquid 18, and second port 40 may be connected to charge the feed liquid 32.

(7) The precipitation bath 16 comprises an open-top container 46 which has an inclined bottom plane 48 for guiding precipitated microparticles 10 to a discharge connection 50 of the extraction means 22.

(8) The horizontally oriented drum 20 is rotatable around its center axis by means of a motor 52, where the output shaft 54 is arranged along the fluid level of the precipitation liquid 18. In this way, the cylindrical mantle of the rotated drum 20 is partially submersed and hence forms a moving surface 56 which is continuously loaded with a revolving film of the precipitation liquid 18.

(9) The system may comprise a central control unit (not shown) for controlling the operating procedure and the process parameters of the various system units.

(10) In use, the inlet 26 of the spraying unit 12 is loaded with a stream 58 of a carrier gas, e.g. air provided from a compressor. Under the effect of the gas stream 58, the feed liquid 32 is atomized into nebular droplets. Advantageously, the size of the droplets is adjusted by the velocity, temperature and humidity of the carrier gas, wherein evaporation leads to a miniaturization of the droplets during flight. In this stage, a fine aerosol jet 60 is generated by non-electric effects.

(11) Then, the jet 60 is ejected through the nozzle 36, where the droplets are electrically charged to a high potential. The nozzle 36 directs the jet 60 against the drum 20, where the charged droplets are attracted by the target electrode 42 and captured in the film of the precipitation liquid 18 coating the mantle or moving surface 56.

(12) The precipitation liquid 18 contains an aqueous solution of an alginate complexing agent including e.g. Ba.sup.2+ ions which permeate the droplets and lead to solidified beads or microparticles 10 containing immobilized enzyme(s).

(13) In addition to the complexing agent, further ingredients may be provided in the precipitation liquid 18 to structurally stabilize the developing beads on their surface. This may be achieved by polymers, specifically polycations, which preferentially adsorb on the surface of the beads and form a complex with the solidifying alginate component thereby providing a stabilizing outer shell.

(14) The solidified beads which sediment on the inclined bottom plane 48 are guided to the discharge connection 50, where a suspension of microparticles 10 can be discharged to the extraction means 22 either in a continuous or a batch mode.

(15) It is also conceivable to separate microparticles of different size by a cation ion exchange process. When the microparticles 10, which were precipitated with ions of comparatively high atomic mass, are fed into a solution of a precipitating ion of lower atomic mass, e.g. Ca.sup.2+ in a saturated CaSO.sub.4-solution, an ion exchange process occurs with the resulting beads having a lower density. As this happens faster on smaller particles having a comparatively larger surface, smaller beads will ascend in the solution, and a separation can be achieved by decantation. At the same time, the ion exchange process leads to a cleaning in the sense of a reduced toxicity of Ca.sup.2+-containing microparticles 10.

(16) The microparticles 10 containing immobilized enzyme(s) are particularly useful in diagnostic test elements designed for glucose tests. Such test elements may be provided on disposable test tapes or test strips, either for optical or electrochemical analyses.

(17) FIG. 2 shows a sectional view of a glucose test element 62 which is generally designed as a two-layered composite on a substrate 62 formed by a transparent plastic carrier. The substrate 62 is coated with a first layer 66 of a reactive test material. This material contains microparticles 10 including immobilized enzymes and some of functional molecules as mediator and dye. The latter reacts by a color change induced by the enzymes which are responsive to glucose. In one exemplary composition, oxidase- or dehydrogenase enzymes are used, e.g. glucose oxidase or glucose dehydrogenase, and the dye includes molybdenum in the form of phosphomolybdic acid.

(18) The first layer 66 is covered by a second layer 68 of test material containing most of the mediator and of the dye which are also present in the first layer. Further, the second layer 68 contains white pigments for separation of a blood sample and for providing a white background for optical measurement of the color change. It is notably important to avoid that enzymes and other functional molecules permeate the optical barrier formed by the second layer 68 specifically during drying of the respective wet chemistry composition. The microparticles 10 immobilize the enzymes in such a way that they cannot reach the second layer 68 during the manufacturing or analysis process.

(19) On the upper side of the test element 62, a spreading web 70 is attached for homogenous and planar distribution of a blood sample. When conducting a diagnostic test, the blood sample is applied by the user as a droplet from a skin wound.