MICROPROBE FOR SELECTIVE ELECTROPORATION AND MANUFACTURING METHOD OF SUCH A MICROPROBE

Abstract

The subject of the invention is a microprobe for selective electroporation comprising at least two metal electrodes (A) immersed in a glass rod (E), characterised in that the glass rod (E) made of a primary glass is 50 μm to 2 mm in diameter, preferably 50 μm to 500 μm, the metal electrodes (A) made of a metal alloy are formed as rods with diameter of 1 μm to 100 μm, preferably 20 μm to 30 μm, wherein endings of those rods are exposed, wherein the primary glass and the metal alloy are matched in such manner that dilatometric softening temperature DTM of the primary glass is highly similar to the temperature of melting for the metal alloy.
The invention also includes a method of manufacturing of such a microprobe.

Claims

1. Microprobe for selective electroporation comprising at least two metal electrodes (A) immersed in a glass rod (E), characterised in that the glass rod (E) made of a primary glass is 50 μm to 2 mm in diameter, preferably 50 μm to 500 μm, the metal electrodes (A) made of a metal alloy are formed as rods with diameter of 1 μm to 100 μm, preferably 20 μm to 30 μm, wherein endings of those rods are exposed, wherein the primary glass and the metal alloy are matched in such manner that dilatometric softening temperature DTM of the primary glass is highly similar to the temperature of melting for the metal alloy.

2. Method according to claim 1, characterised in that the metal alloy exhibits melting temperature lower than dilatometric softening temperature DTM of the primary glass, preferably not more than 50° C. lower, wherein preferably dilatometric softening temperature DTM of the primary glass equals at most 610° C.

3. Microprobe according to claim 1, characterised in that the metal alloy is a silver alloy, preferably silver and tin, for example it is a BAg7(Ag56Sn) alloy.

4. Microprobe according to claim 1, characterised in that it contains at least one air channel (B) for drug delivery preferably 2 μm to 1500 μm in diameter, located inside the glass rod (E).

5. Microprobe according to claim 1, characterised in that it contains at least one optical channel (C) made of a secondary glass for transmission of optical signal, preferably 1 μm to 300 μm in diameter, located inside the glass rod (E).

6. Microprobe according to claim 1, characterised in that it contains at least one imaging channel (D) made of secondary glass for transmission of optical signal, preferably 50 μm to 1500 μm in diameter, located inside the glass rod (E).

7. Method according to claim 6, characterised in that the refractive index nD of the primary glass is lower than refractive index nD of the secondary glass, preferably of least 0.001 lower, and more preferably at least 0.01 lower, and most preferably at least 0.5 lower, for example refractive index nD of primary glass equals 1.51 and refractive index nD of the secondary glass equals at most 2.49.

8. Microprobe according to claim 1, characterised in that the linear thermal expansion coefficient of the primary glass and the secondary glass are similar, wherein preferably for the primary glass in 20-300° C. temperature range linear thermal expansion coefficient is equal to 84.0 10-7K-1, and in 20-450° C. temperature range is equal to 89.0 10-7K-1 or preferably, linear expansion coefficient for the secondary glass in 20-300° C. temperature range is equal to 89.7 10-7K-1, and in 20-450° C. temperature range is equal to 94.5 10-7K-1.

9. Microprobe according to claim 1, characterised in that the primary glass is chosen from the group consisting of SK222, NC-21A, PBG-89, F2 Schott, KB-03 glass or the secondary glass is chosen from the group consisting of Zr3/XV, NC-32, NC-41, LLF1 Schott, F2 Schott, PBG-08 (PBG81), F2/1,67/2, PBS-57A glass.

10. Method of microprobe manufacturing for selective electroporation, especially microprobe according to claim 1, wherein method of thinning is utilized, characterised in that it includes following steps: a) positioning of the rod made of a primary glass with core containing metal alloy in a capillary made of the primary glass, wherein the capillary preferably is generated previously by pulling, b) subjecting of the product from step a) to thinning process in anaerobic atmosphere inside the capillary, c) preparation of a preform comprising: product of step b)in amount of at least 2 units, and rods made of the primary glass, placed together in the tube made of the primary glass, wherein preferably preform from step c) additionally contains at least one optical fibre rod made of a secondary glass, d) treatment of the preform of step c) using the thinning process on a fibre optic drawing tower with pressure control and in protective atmosphere inside the preform, with generation of a glass rod (E) made of the primary glass 50 μm to 2 mm in diameter, preferably 50 μm to 500 μm and, metal electrodes (A) made of a metal alloy formed as rods with diameter of 1 μm to 100 μm, more preferable 20 μm to 30 μm, and the primary glass and the metal alloy are matched in such manner, that dilatometric softening temperature DTM of the primary glass is highly similar to the temperature of melting for the metal alloy.

11. Method according to claim 10, characterised in that after step d) there occurs a process of electrode uncovering on the end of the microprobe, especially using etching method.

12. Method according to claim 11, characterised in that subsequently after the process of electrode uncovering the electrodes of the microprobe are connected to the external power supply by mounting the microprobe to an underlay plate.

13. Method according to claim 11, characterised in that the etching method with a solution of hydrofluoric acid is utilized, wherein preferably when acid to water ratio in solution of hydrofluoric acid equals 1:1, and more preferably, when etching time equals 30 minutes.

14. Method according to claim 10, characterised in that the metal alloy with melting temperature lower than dilatometric softening temperature DTM of the primary glass is utilized, preferably no more than 50° C. lower, wherein preferably the metal alloy is a silver alloy, more preferably silver and tin alloy, for example BAg7 (Ag56Sn) alloy, and dilatometric softening temperature DTM for the primary glass equals 610° C. at most.

15. Method according to claim 10, characterised in that linear thermal expansion coefficient of the primary glass and the secondary glass are similar, wherein preferably for the primary glass in 20-300° C. temperature range linear thermal expansion coefficient is equal to 84.0 10-7K-1, and in 20-450° C. temperature range is equal to 89.0 10-7K-1 or preferably, linear expansion coefficient for the secondary glass in 20-300° C. temperature range is equal to 89.7 10-7K-1, and in 20-450° C. temperature range is equal to 94.5 10-7K-1.

16. Method according to claim 10, characterised in that refractive index nD of the primary glass is lower than refractive index nD of the secondary glass, preferably at least 0.001 lower, and more preferably at least 0.01 lower, and most preferably at least 0.5 lower, for example refractive index nD of the primary glass equals 1.51 and refractive index nD of the secondary glass equals at most 2.49.

17. Method according to claim 10, characterised in that the primary glass is chosen from the group consisting of SK222, NC-21A, PBG-89, F2 Schott, KB-03 glass or the secondary glass is chosen from the group consisting of Zr3/XV, NC-32, NC-41, LLF1 Schott, F2 Schott, PBG-08 (PBG81), F2/1,67/2, PBS-57A glass.

18. Method according to claim 5, characterised in that the refractive index nD of the primary glass is lower than refractive index nD of the secondary glass, preferably of least 0.001 lower, and more preferably at least 0.01 lower, and most preferably at least 0.5 lower, for example refractive index nD of primary glass equals 1.51 and refractive index nD of the secondary glass equals at most 2.49.

Description

PREFERRED EMBODIMENTS OF THE INVENTION

[0039] Invention will be explained closer in the preferred embodiments, with references to the given figures, where:

[0040] FIG. 1 presents microprobe scheme according to the invention, with two metal electrodes A integrated into flexible glass fibre, so called glass rod E,

[0041] FIG. 2 presents microprobe scheme according to the invention with additional functions integrated with microprobe shown on the FIG. 1.

[0042] FIG. 3 presents SEM images of microprobe cross-sections according to the invention with two metal electrodes A,

[0043] FIG. 4 presents preform cross-section for microprobe thinning according to the invention with two metal electrodes A,

[0044] FIG. 5a-b presents view on the glass rods E with metal core obtained by pulling (drawing down),

[0045] FIG. 6 presents scheme of charging for microprobe according to the present invention,

[0046] FIG. 7 presents microprobe according to the invention with a grip enabling connection to the electricity and mechanical adjustment to the sample,

[0047] FIG. 8a-b presents connection by connecting with conductive gluing of E glass rods of microprobe E according to the invention with macroscopic electric wires,

[0048] FIG. 9a-c presents results from CHO-K1—(ATCC® CCL-61™) cells electroporation measurements described in example 3, and

[0049] FIG. 10a-d presents results from H9C2 cells electroporation measurements—cells of rat cardiac muscle described in example 3.

[0050] On the figures following descriptions has been used: A—metal electrode, B—air channel, C—optical channel, D—imaging channel, E—glass rod.

[0051] Following materials have been used for manufacturing of microprobes: [0052] Microprobe design: tubes and rods made of primary glass, i.e. SK222 glass (thermometric type glass made by Krosno Glassworks) [0053] Optical channel C: secondary glass rods, i.e. Zr3/XV glass (designed and melted in ITME) [0054] Metal electrodes A: rods 2 mm in diameter made of BAg7 (Ag56Sn) alloy (manufacturer—Lucas-Milhaupt Gliwice)

[0055] Characteristics of the materials are enlisted in Table 1 and 2:

TABLE-US-00001 TABLE 1 Characteristics of Bag7 alloy used for construction of GM1A microprobes. Characteristic BAg7 alloy Composition [%] Ag 56 Cu: 22 Zn: 17 Sn: 5 Melting temp. [° C.]: 620-650 Density [g/cm.sup.3]  9.4 Stretching resistance [kg/mm.sup.2] 48

TABLE-US-00002 TABLE 2 Characteristics of glass used for construction of GM1A microprobes. Primary Secondary glass glass Characteristic SK222 Zr3/XV Refractive index n.sub.D 1.520 1.609 Linear thermal expansion coefficient for range of: 20-300° C. [10.sup.−7K.sup.−1] 84.0 89.7 20-450° C. [10.sup.−7K.sup.−1] 89.0 94.5 Transformation temperature Tg [° C.] 542 581 Dilatometric softening temperature 610 644 DTM [° C.] Temperatures distinctive for Leitz heating microscope: Temperature of [° C.] curvature 700 680 generation a spherical shape 820 790 generation a semispherical shape 950 865
Linear thermal expansion coefficients for the primary glass and the secondary glass are similar. SK222 glass and Bag7 alloy are chosen due to the alloy melting temperature which is about 50° C. lower than temperature of thinning for the glass. That provides metal liquidity during glass capillary stretching and limits generation of gaps in elongated glass-metal rods. Relatively high thermal expansion coefficient for the primary glass—SK222 glass (89×10.sup.−7K.sup.−1 for 20-450° C. range) lead to the reduction of tensions on glass-metal interface. Zr3/XV secondary glass is matched with primary SK222 glass in terms of refractive index and thermal expansion coefficient making imaging channel D for transmission of optical image with good mechanical properties.
For realisation of present invention it is possible to choose different glass types which are thermally compatible in rheological terms. Below thermally compatible glass pairs with their qualitative-quantitative composition and refractive index n.sub.D are presented:

TABLE-US-00003 NC-21A and NC-32: NC-21A Concentration NC-32 Concentration Composition [% mol] Composition [% mol] SiO.sub.2 56.84 SiO.sub.2 54 B.sub.2O.sub.3 23.19 B.sub.2O.sub.3 21 Al.sub.2O.sub.3 0.61 Al.sub.2O.sub.3 0.5 Li.sub.2O 6.23 Li.sub.2O 5 Na.sub.2O 9.51 Na.sub.2O 8.5 K.sub.2O 3.63 K.sub.2O 3 BaO 5 n.sub.D = 1.5273 n.sub.D = 1.5538 NC-21A and NC-41: NC-21A Concentration NC-41 Concentration Composition [% mol] Composition [% mol] SiO.sub.2 56.84 SiO.sub.2 54.5 B.sub.2O.sub.3 23.19 B.sub.2O.sub.3 22 Al.sub.2O.sub.3 0.61 Al.sub.2O.sub.3 1.5 Li.sub.2O 6.23 Li.sub.2O 5 Na.sub.2O 9.51 Na.sub.2O 8 K.sub.2O 3.63 K.sub.2O 5 PbO 3 BaO 1 n.sub.D = 1.5273 n.sub.D = 1.5374

TABLE-US-00004 NC-21A and LLF1 Schott: n.sub.D = 1.5273 n.sub.D = 1.5481 NC-21A and F2 Schott: n.sub.D = 1.5273 n.sub.D = 1.6200 PBG-89 and PBG-08: PBG-89 Concentration PBG-08 (PBG81) Concentration Composition [% mol] Composition [% mol] SiO.sub.2 45 SiO.sub.2 40 Ga.sub.2O.sub.3 10 Ga.sub.2O.sub.3 13 Bi.sub.2O.sub.3 10 Bi.sub.2O.sub.3 10 PbO 28 PbO 30 CdO 3 CdO 7 ZnO 4 n.sub.D = 1.9060 n.sub.D = 1.9379 F2 Schott and F2/1.67/2 F2/1.67/2 Concentration F2 Schott Composition [% mol] SiO.sub.2 60.7 Al.sub.2O.sub.3 3 PbO 28 K.sub.2O 4 Na.sub.2O 4 As.sub.2O.sub.3 0.3 n.sub.D = 1.6200 n.sub.D = 1.6543 KB-03 and PBS-57A KB-03 Concentration PBS-57A Concentration Composition [% mol] Composition [% mol] B.sub.2O.sub.3 62.97 SiO2 53.10 ZnO 6.57 PbO 44.20 CaO 9.53 Al.sub.2O.sub.3 0.65 Na.sub.2O 16.16 Na.sub.2O 0.86 NaF 4.77 K.sub.2O 0.85 As.sub.2O.sub.3 0.33 n.sub.D = 1.5415 n.sub.D = 1.8467

Example 1

Microprobe for Electroporation

[0056] In present embodiment microprobe for electroporation presented on scheme FIG. 1 and on the SEM image on FIG. 3, is a glass rod E with two integrated metal electrodes A. Alternatively, microprobe contains higher amount of metal electrodes A, what is depicted on FIG. 2, being in form of microwires, and immersed in glass rod E. Continuous and long metal electrodes A exhibit small diameters equal to 29 μm and 28 μm, respectively, and distance between them equals to 37 μm. External diameter of glass rod equals to 458 μm.

Depending on the additional application microprobes are integrated in one unit also with other elements such as imaging channel D for transmission of optical image, optical channel C for transmission of optical signal or air channel B for drug delivery.

Example 2

Manufacturing of GM1A Type Microprobe Containing Optical Channel C for Transmission of Optical Image

[0057] Process for microprobe manufacturing is performed in few steps: [0058] Manufacturing of capillaries from SK222 glass with 2 mm internal diameter and internal/external diameter ratio equal to about 0.5. For this purpose from φext/φint 15/11 tubes φext 10 were pulled out and after combination with φext 15 final capillaries were pulled out with 4.2/2 φext/φint dimensions. [0059] Pulling of E glass rods with metal core. [0060] Rod made of BAg7 alloy after polishing and defatting was placed in immersed capillary made of SK222 glass. Capillary was pumped out and flushed with argon few times before thinning in order to remove oxygen and to avoid metal oxidation. During thinning process anaerobic conditions were sustained inside the capillary (argon). Rods were drawn down (with metal core) with 0.3-1.1 external diameter, presented on FIG. 2. [0061] Pulling of C glass rods for transmission of optical image. [0062] Optical fibre structure rods were manufactured using “rod-tube” method by placement of rod made of Zr3/XV glass in tube made of SK222 glass and subsequently by thinning of preform to the 0.7 mm diameter. [0063] Pulling of SK222 rods. [0064] Preparation of preform [0065] Preform structure for probe pulling consisted of two glass-metal rods of 0.7 mm diameter, optical fibre rod placed between them of 0.7 mm in diameter and SK222 glass rods. Whole set was placed in tube made of SK222 glass. [0066] Probes thinning (pulling).

[0067] From prepared preform rods were pulled with internal 0.3-0.5 mm diameter with anaerobic atmosphere inside preform. As a result of the above process microprobe was prepared, which is illustrated on FIG. 3, 350 μm in diameter and containing metal electrodes A with 20 μm in diameter. Optical channel C for transmission of optical image is not placed exactly between electrodes, and its translocation happened in the step of preform thinning and is irrelevant for functionality of microprobe manufactured in this manner. Few metal electrode Only few discontinuities of metal electrodes were found in pulled probes. During selection over a dozen of probes 10 to 30 cm long containing continuous metal rods were chosen and selected for further application studies. Parameters of manufactured microprobes are enlisted below in Table 3.

TABLE-US-00005 TABLE 3 Geometric parameters of GM1A probes. Geometric parameters of GM1A probes GM1A/2 GM1A/3 Diameter of glass rod E [μm] 458  352  Diameters of metal electrodes A [μm] 29 and 28 21 and 20 Distance between metal electrodes A - 37 31 [μm] Diameter of optical channel [μm] 28 23
Manufactured microprobes were connected to the external power supply by mounting of microprobes to the plates including mounting of metal electrodes A to the standard electric wires. Mounting of microprobe to the underlay plate was performed using microscope and micromanipulators. Epoxy glue was used. In the next step microelectrodes were connected with microscopic electric wires using Epo-Tek conductive adhesive. Microprobe was heated in oven in 300° C. temp. for 15 min. In these conditions glue was hardened. Bonding of A metal electrodes with macroscopic electric wires is depicted on FIG. 8. Rigid microprobe endings were obtained in this way enabling connection to electricity and to microprobe manipulation during experiment. Manufacturing of microprobe electrical connections shown on FIG. 7 enabled resistance measurement for metal electrodes A and measurement of breakdown voltage listed below in Table 4. Measurement was carried out under microscope because of need for metal electrode A length measurement and need for precise power connection.

TABLE-US-00006 TABLE 4 Summary of results for electrode resistance measurements in GM1A microprobe. A metal electrode length (from connector to connector) Resistance [Ω] (10.sup.−3)[mm] 33.8 117.98 34 118.48 70.8 154.72 41.4 154.87 31.6 98.13 28.3 96.27 86.5 97.03 70 94.99
Obtained results show low resistance of metal electrodes A what enables generation difference of potentials between two metal electrodes A without existence of unfavourable heat conditions in microprobe. It is also possible to utilize short electric pulses used in a few electroporation techniques.
Obtained results indicate that microprobe has electrodes with homogeneous diameter inside E glass rod. Subsequently, experiments were conducted leading to define breakdown voltage. Tests were conducted in aqueous solution of sodium chloride from zero concentration to obtaining of saturated solution in room temperature (26.5% concentration). Laboratory power adapter with regulated voltage in 0-30 V range was utilized to measure breakdown voltage. Performed tests have given negative results what means that breakdown voltage was higher than 30 V. That enables unconstrained usage of microprobe in aqueous environment (typical for laboratory cell cultures) without worries about breakdown and destruction of samples or microprobe.

Example 3

Electroporation Using Manufactured Microprobe According to the Invention

[0068] Microprobes with 10-30 cm length and 350 μm in diameter with two metal electrodes A with about 20 μm in diameter, illustrated on FIG. 1, were manufactured. Metal electrodes A were exposed using etching with a solution of hydrofluoric acid, wherein acid to water ration in hydrofluoric acid was 1:1, and etching process lasted for 30 minutes. Resistance for measured microprobes 20-25 cm in length was equal to 30-80Ω. Tests performed in salt solution shown that breakdown voltage was higher than 30 V. Electroporation tests using manufactured microprobes were performed for opening of cell membrane and delivery of substances into the cells. 3-5 V voltage was applied to the microprobes. Two cell lines were used for studies: CHO-K1 cells—(ATCC® CCL-61™)—results of the studies were presented on FIG. 9a-c and H9C2 rat cardiac muscle cells were used. To verify the occurrence of cell electroporation phenomenon marker method using Trypan Blue was utilized. In cases of both cell lines it was unequivocally confirmed that electroporation is occurring in single cells in active range of microprobe. Migration of substances from the solution to the cell interior is an indication of occurrence of electroporation. It is possible to observe due to accumulation of marker in the cell which stains it blue. On FIG. 6a-c measurement results of CHO-K1 (ATCC® CCL-61™) cells electroporation were presented. During the experiment the distance between metal electrode A was equal to 226.93 μm, between its internal edges, and 277.83 μm between its external walls, electrode thickness was equal to 28.62 μm, and voltage and current intensity were respectively equal to: 1.7-2 V, 1.9 μA. Cells after electroporation (higher electrode) were stained with trypan blue and exhibited flattened morphology, what is illustrated on FIG. 9b. Cells placed in 300 micron distance from electrodes during the experiment (FIG. 9c) have not been electroporated and were left intact (so called control cells). Results of the electroporation experiment for rat cardiac muscle cell line—H9C2 were depicted on FIG. 10a-d. During the experiment distance between metal electrode A was equal to 156.46 μm, between its internal edges, and 207.83 μm between its external walls, metal electrode A thickness was equal to 27.11 μm, and voltage and current intensity were respectively equal to: 1.7-2 V, 1.9 μA. FIG. 10c shows a cell after electroporation near upper electrode in which a stained nucleus is visible. Cells which did not undergo electroporation (so called control cells) show no sings of staining according to the FIG. 10d.