MEDICAL PROTO MICROELECTRODE, METHOD FOR ITS MANUFACTURE, AND USE THEREOF

Abstract

A proto-microelectrode, a proto-microelectrode bundle and array, a method of manufacture of the proto-microelectrode, and a method of using the proto-microelectrode, the proto-microelectrode being capable of forming a microelectrode upon implantation into soft tissue, and includes an oblong electrode body; an optional first coat of electrically non-conducting material on the electrode body; a second coat of water insoluble flexible polymer material enclosing, at a distance, the electrode body and the first coat, the second coat including one or more through openings; a first layer of ice disposed between the electrode body and the second coat.

Claims

1. Proto-microelectrode capable of forming, upon insertion into soft tissue, a microelectrode, the proto-microelectrode comprising or consisting of: an oblong electrode body of electrically conducting material having a front (distal) end and a rear (proximal) end, the electrode body comprising or consisting of metal or metal alloy or an electrically conducting form of carbon or an electrically conducting polymer or a combination thereof; an optional first coat of electrically non-conducting material on the electrode body extending along it from its rear end towards its front end, the electrode body comprising one or more sections not covered by the first coat; a second coat of water insoluble flexible polymer material disposed at a distance from and enclosing the electrode body and, if present, the first coat or a portion thereof, the second coat comprising one or more through openings or windows; a first layer of ice (frozen aqueous solution) optionally comprising pharmacologically active agent disposed between the electrode body and the second coat.

2. The proto-microelectrode of claim 1, wherein the electrode body is flexible, in particular resiliently flexible, or stiff.

3. The proto-microelectrode of claim 1, wherein the first layer of ice optionally comprising biologically active agent has a melting point of from −5° C., more preferred of from −3° C. or −2° C., in particular from −1,5° C., preferably from −1,0° C., particularly preferred from −0,8° C. or −0,7° C., or even from −0,5° C. to 0° C.

4. The proto-microelectrode of claim 1, wherein one or more of said one or more through openings are disposed in distal portion(s) of the second coat.

5. The proto-microelectrode of claim 4, wherein one or more of said one or more through openings, in particular all through openings, are disposed in a portion of the second coat extending from half of its length to the distal end, in particular from two thirds or three fourths of its length to the distal end, most preferred in a portion extending in a proximal direction from the distal end over a distance of 5 percent or 10 percent of the length of the electrode body.

6. The proto-microelectrode of claim 4, wherein one or more of said one or more through openings, in particular all through openings, are disposed in a portion of the second coat extending from half of its length to the proximal end, in particular from two thirds or three fourths of its length to the proximal end, most preferred in a portion extending in a distal direction from the proximal end over a distance of 5 percent or 10 percent of the length of the electrode body.

7. The proto-microelectrode of claim 1, wherein second coat has a wall thickness that is smaller than the diameter of the electrode body or the diameter of the combination of electrode body and first coat, in particular has a thickness of less than 50%, preferably of less than 30%, most preferred of less than 15% or 10% of said diameters.

8. The proto-microelectrode of claim 7, wherein the wall thickness of the second coat is up to 20 μm, in particular is from 2 μm to 5 μm.

9. The proto-microelectrode of claim 1, wherein the diameter of the electrode body is from 1 μm to 100 μm or more, in particular from 2 μm to 10 μm or 25 μm or 40 μm.

10. The proto-microelectrode of claim 1, wherein a portion of the second coat has the form of a bellows tube.

11. The proto-microelectrode of claim 1, comprising an electrical lead attached to a proximal portion of the electrode body, wherein the second coat extends to and encloses a distal portion of the lead.

12. The proto-microelectrode of claim 1, comprising a second layer of ice optionally comprising pharmacologically active agent disposed on the second coat.

13. The proto-microelectrode of claim 1, wherein the ice (frozen aqueous solution) comprises any combination of ammonium, calcium, iron, magnesium, potassium, quaternary ammonium, sodium, copper, acetate, carbonate, chloride, citrate, fluoride, nitrate, nitrite, oxide, phosphate, and sulfate.

14. The proto-microelectrode of claim 1, wherein the ice (frozen aqueous solution comprises a buffer capable of regulating the pH of the aqueous solution prior to freezing between 6.5 up to 7.5.

15. The proto-microelectrode of claim 1, wherein the electrode body is chemically modified to be used in voltammetry based methods.

16. A method of generating a micro-electrode disposed in soft tissue comprising inserting the proto-microelectrode of claim 1, into the tissue, wherein the proto-microelectrode has a temperature at the start of insertion of below 0° C., in particular of below −1° C. or −2° C., preferably of below −5° C.

17. Use of the proto-microelectrode of claim 1 for implantation into soft tissue.

18. Use of the of the proto-microelectrode of claim 1 for monitoring electrochemical signals.

19. Method of manufacture of a proto-microelectrode for insertion into soft tissue, comprising: providing a first pre-stage microelectrode comprising or consisting of an oblong electrode body of electrically conducting material having a front (distal) end and a rear (proximal) end; optionally comprising a first coat of electrically non-conducting material on the electrode body extending along it from its rear end towards its front end, the electrode body comprising one or more sections not covered by the first coat; a second coat of water insoluble flexible polymer material disposed at a distance from and enclosing the electrode body or a portion thereof, the second coat comprising one or more through openings; a layer of porous carbohydrate material disposed between the electrode body and the second coat; providing a second pre-stage microelectrode by substituting the layer of porous carbohydrate material by water optionally comprising pharmacologically active agent; cooling the thus transformed second pre-stage microelectrode to a temperature capable of transforming the layer of water optionally comprising pharmacologically active agent to a first layer of ice for a time sufficient for complete transformation.

20. The method of claim 19, comprising: optionally providing the second coat with a layer of gelatin; providing the second coat or, if present, the layer of gelatin on the second coat with a second layer of ice optionally comprising pharmacologically active agent.

21. The method of claim 19, wherein the first pre-stage microelectrode comprises a flexible electrical lead attached to the proximal end of the electrode body and wherein the second coat encloses a distal terminal portion of the flexible electrical lead.

22. The method of claim 19, wherein the layer of porous carbohydrate material on the electrode body is formed by providing an aqueous solution comprising or consisting of water and more than 20% by weight of glucose and/or other mono- or disaccharide of high solubility in water or a combination thereof, in particular of more than 40% or 45% by weight; providing a form comprising a channel of cylindrical form or other rotationally symmetric form closed at its one end; disposing the electrode body with its distal end foremost in the channel; filling the channel up to a desired proximal level of the electrode body with said aqueous solution; cooling the form to a freezing temperature of the aqueous solution; separating the electrode body with adhering frozen aqueous solution from the form and disposing it either, while keeping it frozen, in an low-pressure environment for a time sufficient to transform the frozen aqueous solution to said layer of porous carbohydrate, wherein a pressure in the low-pressure environment is below 1000 Pa, in particular below 500 Pa or 200 Pa, or placing the container 4 with its contents in an oven, heating the container with its contents to a temperature above room temperature, in particular to a temperature of 50° C. or more, until the aqueous solution has been transformed to a caramelized first carbohydrate layer on the electrode body and the assembly to a second pre-stage microelectrode.

23. The method of claim 22, wherein the form is separable in a plane in which the cylinder axis or the axis of a channel of other rotationally symmetric form is disposed.

24. Proto-microelectrode array comprising two or more proto-microelectrodes according to claim 1 joined at their proximal portions by an array base.

25. Proto-microelectrode bundle comprising two or more proto-microelectrodes according to claim 1 and a bundling element, in particular of annular form, enclosing them at their proximal portions.

Description

SHORT DESCRIPTION OF THE FIGURES

[0059] Except for FIGS. 2a and 18, all figures are longitudinal sections of proto-microelectrodes or their pre-stages and of containers used in their production.

[0060] FIGS. 1 through 16 illustrate the manufacture of a first embodiment of the proto-microelectrode of the invention; and the proto-microelectrode

[0061] FIGS. 1, 1a illustrate a cylindrical (central axis A-A), partially insulated microelectrode body for use in the invention, in a sectional view (FIG. 1), and an enlarged partial sectional view of its terminal proximal portion (FIG. 1a), the insulation extending proximally of the microelectrode body to cover and insulated a flexible electrically conducting lead connecting the microelectrode body with an apparatus for electrode control (not shown);

[0062] FIG. 2 shows, in a longitudinal section (axis C-C), a first container consisting of two halves kept together by an annular retainer and comprising a cylindrical channel closed its bottom end;

[0063] FIG. 2a is a top view in a distal direction of the electrode body inserted into the first container;

[0064] FIG. 3 shows the insulated microelectrode body of FIG. 1 inserted into the first container of FIG. 2 in a manner to make their axes A-A and C-C coincide;

[0065] FIG. 4 shows the void between the electrode body and the inner wall of the first container filled with a concentrated aqueous solution of glucose;

[0066] FIG. 5 shows the aqueous solution of glucose in the void of FIG. 4 in a frozen state;

[0067] FIG. 6 shows a first pre-stage of the electrode of the invention formed by removing the electrode body with the frozen solution adhering to its lateral and distal faces from the first container;

[0068] FIG. 7 shows a second pre-stage of the electrode of the invention of which the electrode body is covered with a porous layer of dry glucose formed by freeze-drying of the first pre-stage's frozen glucose solution;

[0069] FIG. 8 shows a third pre-stage of the electrode of the invention comprising a coat of water-insoluble flexible polymer disposed on the external face of the layer of porous dry glucose, the polymer layer having been formed by gaseous deposition of polymer precursors;

[0070] FIG. 9 shows the third pre-stage of FIG. 8 provided with a lateral opening or window in the coat of water-insoluble flexible polymer;

[0071] FIG. 10 shows the modified third pre-stage of FIG. 9 inserted into the cylindrical void of a second container in a manner making their axes A, D coincide;

[0072] FIG. 11 shows the modified third pre-stage of FIG. 10 immediately upon filling the void between the third pre-stage and the inner wall of the second container with water optionally comprising small amounts of soluble additives such as pharmaceuticals and vitamins;

[0073] FIG. 12 illustrates the action of water optionally comprising small amounts of soluble material on the third pre-stage in the second container upon storing for a time causing dissolution of the porous glucose layer;

[0074] FIG. 13 illustrates the substitution of the aqueous solution of glucose inside of the layer of flexible, water insoluble polymer material of the third pre-stage by diffusion/convection of water optionally containing small amounts of soluble material added to and withdrawn from the container continuously or discontinuously;

[0075] FIG. 14 shows the third pre-stage of the proto-microelectrode being withdrawn from the second container while cooling by a flow of cold gas directed at the portion of the third pre-stage emanating from the second container;

[0076] FIG. 15 shows the proto-microelectrode of the invention formed from the third pre-stage of during its withdrawal from the second container;

[0077] FIG. 16 shows a first variety of the proto-microelectrode of the invention with its coat of polymer material covered by a thin layer of ice;

[0078] FIGS. 17 through 32 illustrate the manufacture of a second embodiment of the proto-microelectrode of the invention; and the proto-microelectrode.

[0079] FIG. 17 shows a cylindrical (central axis A′-A′) microelectrode body lacking electrical insulation for use in the invention to the distal terminal portion of which a flexible electrically conducting lead is connected to provide for electrical connection of the microelectrode body with an apparatus for electrode control (not shown);

[0080] FIGS. 18, 19 show the microelectrode body of FIG. 17 inserted into the first container of FIG. 2 in a manner to make their axes A′-A′ and C-C coincide;

[0081] FIG. 20 shows the void between the electrode body and the inner wall of the first container filled with a concentrated aqueous solution of glucose;

[0082] FIG. 21 shows the aqueous solution of glucose in the void of FIG. 20 in a frozen state;

[0083] FIG. 22 shows a first pre-stage of the electrode of the invention formed by removing the electrode body with the frozen solution adhering to its lateral and distal faces from the first container;

[0084] FIG. 23 shows a second pre-stage of the electrode of the invention of which the electrode body is covered with a porous layer of dry glucose formed by freeze-drying of the first pre-stage's frozen glucose solution;

[0085] FIG. 24 shows a third pre-stage of the electrode of the invention comprising a coat of water-insoluble flexible polymer disposed on the external face of the layer of porous dry glucose, the polymer layer having been formed by gaseous deposition of polymer precursors. The coat water-insoluble flexible polymer extends proximally of the electrode body to cover and thereby isolate the flexible electrically conducting lead;

[0086] FIG. 25 shows the third pre-stage of FIG. 8 provided with a lateral opening or window in the coat of water-insoluble flexible polymer;

[0087] FIG. 26 shows the modified third pre-stage of FIG. 25 inserted into the cylindrical void of the second container in a manner making their axes A′, D coincide;

[0088] FIG. 27 shows the modified third pre-stage of FIG. 26 immediately upon filling the void between the third pre-stage and the inner wall of the second container with water optionally comprising small amounts of soluble additives such as pharmaceuticals and vitamins;

[0089] FIG. 28 illustrates the action of water optionally comprising small amounts of soluble material on the third pre-stage in the second container upon storing for a time causing dissolution of the porous glucose layer;

[0090] FIG. 29 illustrates the substitution of the aqueous solution of glucose inside of the layer of flexible, water insoluble polymer material of the third pre-stage by diffusion/convection of water optionally containing small amounts of soluble material added to and withdrawn from the container continuously or discontinuously;

[0091] FIG. 30 shows the third pre-stage of the proto-microelectrode being withdrawn from the second container while cooling by a flow of cold gas directed at the portion of the third pre-stage emanating from the second container;

[0092] FIG. 31 shows the proto-microelectrode of the invention formed from the third pre-stage of during its withdrawal from the second container;

[0093] FIG. 32 shows a first variety of the proto-microelectrode of the invention with its coat of polymer material covered by a thin layer of ice.

DESCRIPTION OF PREFERRED EMBODIMENTS

Example 1. Manufacture of a First Embodiment of the Proto-Microelectrode of the Invention

[0094] An oblong cylindrical microelectrode body 1 of an electrically conducting material such as, for instance, copper, silver, gold or platinum, having a central axis A-A is covered by a coat 2 of an insulating material, in particular of a polymer material, extending from its rear (proximal) terminal portion 1b towards its front (distal) end in a manner so as to leave a distal terminal portion 1a of the electrode body 1 uncovered (FIG. 1a). Attached to the proximal end of the proximal terminal portion 1b by solder 7 is a flexible metallic wire 8 covered with an insulating coat 3 of lacquer or polymer material (FIG. 1b). At its other end, the wire 8 is connected to an apparatus (not shown) for electrical control of and/or receipt of electrical signals from the electrode body 1. In a variety of the microelectrode of the invention illustrated in FIG. 17 and its various pre-stages (not shown), the coat 2 of insulating material can extend proximally of the electrode body 1 for insulation of the flexible metallic wire 8, thereby making the separate insulation layer 3 redundant.

[0095] The electrically insulated rear or proximal terminal portion 1b of the partially insulated electrode body 1, 2 is shown in FIG. 1a enlarged and in greater detail. It comprises, at a short distance from its proximal end, an annular bulge 9 allowing arms 9′, 9″ of a pincer-like gripping device to hold it for manipulation. For the sake of reducing visual complexity in the drawing, the annular bulge 9 comprised by all microelectrode bodies 1 of FIGS. 3 through 13 is only shown in FIG. 1a.

[0096] A first container 4 comprises a cylindrical channel 5 having an upper open end and a bottom end 6 of rounded form, such as of hemi-circular or hemi-elliptical form, is shown in FIG. 2 in an axial section B-B (FIG. 2a). The first container 4 is of a low-friction material such as polypropylene, polyfluorinated polyalkene or silicone, or has the face of its channel 5 covered with a layer of such material. It comprises two symmetrical halves 4a, 4b releaseably joined by an annular retainer 25 at their mutually abutting flat faces extending in the longitudinal direction of the container.

[0097] The partially insulated microelectrode body 1, 2 is inserted into the cylindrical channel 5 with its distal end 1a foremost (direction R) in a centered manner to make its axis A-A coincide with the channel axis C-C. The insertion is to a depth in the vicinity of the hemi-circular bottom or until the front end of the distal portion 1a is contacting the bottom 6, as shown in FIG. 3. Alternatively, its distal end can be disposed at a short distance from the bottom 6 (not shown).

[0098] Upon insertion of the partially insulated microelectrode body 1, 2, the remaining void of channel 5 is filled with an aqueous solution 11 comprising 45% by weight of glucose up to about the height of the insulated proximal terminal microelectrode body portion 1b (FIG. 4).

[0099] Next, the first container 4 with its contents 1, 2, 11 is cooled to a sufficiently low temperature, such as to a temperature of −15° C. or −20° C., thereby transforming it into a frozen state 11′ (FIG. 5) to make the contents 1, 2, 11′ in combination with the distal terminal portion 1b of the electrode body 1 and the insulated 8 electrical lead 3 constitute the in-situ first pre-stage of the microelectrode of the invention. The contents 1, 2, 11′ are then withdrawn from the first container 4 by releasing the retainer 25 to isolate the first pre-stage 10 of the microelectrode of the invention (FIG. 6).

[0100] While the first pre-stage 10 is kept at a low temperature to prevent the frozen glucose solution 11′ from thawing, it is exposed to low pressure, such as a pressure of 0.1 mm Hg or 0.01 mm Hg or lower, for a sufficient period of time to transform its frozen glucose layer 11′ to a porous layer 12 of dry glucose. The so produced second pre-stage 10 a of the microelectrode of the invention is shown in FIG. 7.

[0101] Alternatively the container 4 with the partially insulated electrode body 1, 2 covered by frozen aqueous carbohydrate solution 11′ is placed in an oven and heated to a temperature above room temperature, in particular to a temperature of 50° C. or more, until the aqueous solution has been transformed to a caramelized first carbohydrate layer on the electrode body 1, 2 and the assembly thus to a second pre-stage microelectrode.

[0102] Next, the distal and lateral face of the porous glucose layer 12 of the second pre-stage 10 a is covered with a silicone polymer coat 13 by dip coating (FIG. 8). The thickness of the polymer coat 12 of the thus produced third pre-stage 10b of the microelectrode of the invention is from about 2 μm to about 5 μm.

[0103] In the following step one or more openings or windows 14 are provided in a distal portion of the polymer coat 13 by removing portions of the coat 13 by a micro-diamond knife or by evaporation by laser means to form the fourth pre-stage 10c of the microelectrode of the invention (FIG. 9). When using laser means it is advantageous to evaporate the polymer step-by-step to provide an opening with a smooth contour.

[0104] In the first of the final production steps of the proto-microelectrode 20 of the invention (cf. FIG. 15), the fourth pre-stage 10c is disposed in the cylindrical void 18 of a second container 15 of, for instance, glass or metal, with its distal end foremost and centered in respect of the container axis D-D (FIG. 10).

[0105] Next, the void 18 is filled up to the proximal terminal portion 1b of the fourth pre-stage's 10c (cf. FIG. 10) microelectrode body 1 with water 16 optionally comprising small amounts of biologically active agent, such as anti-coagulation agent, antibacterial agent, anti-virus agent osmotic pressure controlling agent (FIG. 11); the use of artificially prepared spinal fluid or physiological aqueous solutions for this purpose is recommended.

[0106] FIG. 12 illustrates a production stage at which the water or aqueous solution 16 surrounding the fourth pre-stage 10c (cf. FIG. 10) has entered through window 14 the space enclosed by the polymer coat 13 containing porous glucose 11′, which is dissolved to form a diluted aqueous solution 16′ of glucose optionally containing said small amounts of biologically active agent(s).

[0107] Further, in particular continuous, provision of water 16 or a diluted aqueous solution 16 optionally containing small amounts of biologically active agent(s) supplied to the second container 2 by one or more feeding tube(s) 19 and draining the liquid contents 16′ of the container by one or more draining 19′ tubes, results in the aqueous fluid 16′ originally present in the second container 2 to be substituted by water 16 optionally containing small amounts of biologically active agent (FIG. 13).

[0108] In the final production step the so formed in-situ fifth pre-stage 10d of the proto-microelectrode of the invention is slowly withdrawn (in axial direction S) from the second container 2 (FIG. 14) while cooling it with a stream of cold gas G at a temperature of −20° C. or lower, such as with air or nitrogen or carbon dioxide formed from dry ice, fed by tubes 23, 23′ with their outlet openings pointing towards pre-stage 10d emerging from the second container 2, successively (in parallel with removal) transforming the water or aqueous solution 16 disposed inside of the flexible polymer coat 13 to ice 16′. In this manner pre-stage 10d is transformed to the proto-microelectrode of the invention 20 (FIG. 15) of which the entire space between the partially insulated electrode body 1, 2 and the coat 13 of flexible polymer material is filled with ice 16 optionally comprising small amounts of biologically active agent.

[0109] The first variety 20a of the proto-microelectrode 20 of the invention shown in FIG. 16 additionally comprises a second layer 17 of ice optionally comprising biologically active agent(s) on the distal and lateral portions of the electrode 20 covered by the flexible polymer layer 13 and including the window 14.

[0110] FIG. 16 shows a second variety 21 of the proto-microelectrode of the invention in which the flexible polymer coat 13′ disposed on the first layer of ice 16′ optionally comprising small amount(s) of biologically active agent(s) extends distally of the distal terminal portion 1b of the electrode body 1 so as to cover the flexible electrical lead 8, which thus does not require separate insulation. In the production of variety 21 the polymer coat 13′ (to be later provided with opening(s) or window(s) 14′) is applied to the second pre-stage 10a instead of the polymer coat 13 (FIG. 7). Since being substantially identical with corresponding production stages resulting in the proto-microelectrode of the invention 20, the corresponding stages of manufacture of the second variety 21 of the proto-microelectrode of the invention intermediate between those shown in FIGS. 5 and 15 are not separately illustrated.

[0111] FIG. 18 shows a second variety 22 of the proto-microelectrode of the invention differing from the first variety 21 by lacking insulation 2 on the electrode body 1 while sharing all other features.

Example 2. Manufacture of a Second Embodiment of the Proto-Microelectrode of the Invention

[0112] Attached by solder 107 to the proximal terminal portion 101b of an oblong cylindrical microelectrode body 101 of an electrically conducting material such as, for instance, copper, silver, gold or platinum, having a central axis A′-A′ is a flexible metallic wire 108 (FIG. 17). At its other end, the wire 108 is connected to an apparatus (not shown) for electrical control of and/or receipt of electrical signals from the electrode body 101.

[0113] Upon insertion of the microelectrode body 101 into the cylindrical channel 5 of the first container 4a, 4b of FIG. 19 with its distal end 101a foremost and in a centered manner to make its axis A′-A′ coincide with the channel axis C-C and to a depth in the vicinity of the hemi-circular bottom or until the front end of the distal portion 101a is contacting the bottom 6 (FIGS. 19, 20, 21), the remaining void of the first container 4a, 4b is filled with an aqueous solution 111 comprising 45% by weight of glucose to about the height of the proximal terminal microelectrode body portion 101b (FIG. 20).

[0114] Next, the first container 4 with its contents 101, 111 is cooled to a low temperature, such as to a temperature of −15° C. or −20° C., sufficient for transforming the aqueous glucose solution 111 into a frozen state 111′ (FIG. 21), thereby making the contents 101, 111′ in combination with the proximal terminal portion 101b of the electrode body 101 not covered by frozen glucose solution 111′ and the electrical lead 108 constitute a direct precursor of the first pre-stage of the microelectrode of the invention (FIG. 21). The contents 101, 111′ are then removed from the first container 4 by releasing the retainer 25 to provide the first pre-stage 110 of the microelectrode of the invention (FIG. 22).

[0115] While the first pre-stage 110 is kept at a low temperature to prevent the frozen glucose solution 111′ from thawing, it is exposed to a low pressure, such as a pressure of 0.1 mm Hg or 0.01 mm Hg or lower, for a sufficient period of time to transform its frozen glucose layer 111′ to a porous layer 112 of dry glucose. The so produced second pre-stage 110a of the microelectrode of the invention is shown in FIG. 23. Alternatively, the container 4 with the electrode body 101 covered by frozen aqueous carbohydrate solution 111′ is placed in an oven and heated to a temperature above room temperature, in particular to a temperature of 50° C. or more, until the aqueous solution has been transformed to a caramelized first carbohydrate layer on the electrode body 1 and the assembly thus to a second pre-stage microelectrode.

[0116] Next, the distal and lateral face of the porous glucose layer 112 of the second pre-stage 110a is covered up to its proximal end with a polymer coat 113 of Parylene C by evaporation and deposition of Parylene C precursors at low pressure (FIG. 24). The thickness of the polymer coat 113 of the thus produced third pre-stage 100b of the microelectrode of the invention is from about 2 μm to about 5 μm. The Parylene C coat then further extends proximally to cover, thereby electrically isolating the connecting wire or lead 108.

[0117] In the following step one or more openings or windows 114 are provided in a distal portion of the polymer coat 113 by removing portions of the coat 113 by a micro-diamond knife or by evaporation by laser means to form the fourth pre-stage 110c of the microelectrode of the invention (FIG. 25).

[0118] In the first of the final production steps of the second embodiment of the proto-microelectrode 120 of the invention, the fourth pre-stage 110c is disposed in the cylindrical void 18 of the second container 15 of FIG. 26.

[0119] Next, the void 18 with the fourth pre-stage electrode 110c is filled up to the proximal terminal portion 101b (FIG. 27) with water 16.

[0120] By successively dissolving the porous glucose layer 112 (by water or aqueous solution comprising biologically active agents) added water 16 or aqueous solution 16 containing small amounts of biologically active agent(s) then enters through window 114 the space enclosed by the polymer coat 113 to form an aqueous solution 16′ of glucose optionally containing small amounts of biologically active agent, such as anti-coagulation agent, antibacterial agent, anti-virus agent osmotic pressure controlling agent (FIG. 28). Further, in particular continuous, supply of water 116 optionally containing small amounts of biologically active agent(s) to the second container 15 by one or more feeding tube(s) 119 and draining the liquid contents 116′ of the container by one or more draining 119′ tubes results in the aqueous fluid 16′ originally present in the second container 15 to be substituted by water 16 optionally containing small amounts of biologically active agent (FIG. 29).

[0121] In the final production step the so formed in-situ fifth pre-stage 110d of the second embodiment of the proto-microelectrode of the invention is slowly withdrawn (in axial direction S) from the second container 15 (FIG. 30) while cooling it with a stream of cold gas G at a temperature of −20° C. or lower, such as with air or nitrogen or carbon dioxide formed from dry ice, fed by tubes 23, 23′ with their outlet openings pointing towards pre-stage 110d emerging from the second container 15, successively (in parallel with removal) transforming the water or aqueous solution 16 disposed inside of the flexible polymer coat 113 to a layer of ice 118. In this manner pre-stage 110d is transformed to the second embodiment of the proto-microelectrode of the invention 120 (FIG. 31) of which the entire space between the electrode body 101 and the coat 113 of flexible polymer material is filled with ice 118, optionally comprising small amounts of biologically active agent.

[0122] A first variety 121 of the proto-microelectrode 120 of the invention is shown in FIG. 32. It comprises additionally a second layer 117 of ice optionally comprising biologically active agent(s) on the distal and lateral portions of the flexible polymer layer 113 and on the portion of the ice 116′ layer at window 114 not covered by layer 113.