Biochip electroporator and its use in multi-site, single-cell electroporation

• US 20070155015A1
• Filed: 01/29/2004
• Published: 07/05/2007
• Est. Priority Date: 01/29/2004
• Status: Active Grant

• Current Assignee: Narvalus S.r.l.
• Original Assignee: Narvalus S.r.l.
• Inventors: Cellere, Giorgio, Vassanelli, Stefano
• Granted Patent: US 8,017,367 B2
• Time in Patent Office: Days
• US Class Current: 435/461
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Abstract

The introduction of genetic material or molecules of biological interest into cells is a procedure with an increasing interest both for experimental and application purposes, so that electroporation is a widely used technique, but the electroporation of single adhering cells is still impaired. The present application describes an apparatus for the electroporation of any kind of cell adhering to a substrate at any stage of development, where an electrical signal can be driven and applied to a single adhering cell in culture in order to obtain its electroporation. The method to electroporate a single adhering cell with the apparatus of the invention is also described.

Claims

1. Apparatus for electroporation comprising a wave generator, a biochip containing an array of microelectrodes and a control system that permits to transfer of a signal to a pre-selected single microelectrode of the biochip.

2. Apparatus according to claim 1 wherein the control system consists of a personal computer equipped with a software program capable of designing various waveform signals and a switching system controlling the wave generator output.

3. Apparatus according to claims 1 wherein the biochip comprises an array of microelectrodes of a size comparable to a the cell to be electroporated and each of said microelectrodes being driven separately from the others allowing very precise and punctual control of the electroporation process.

16. A method of electroporation wherein an apparatus according to claims 1 is used.

17. The method according to claim 16 wherein said apparatus performs one or more electroporations to at least a single adhering cell.

18. The method according to claim 17 wherein said apparatus comprises a biochip according to claims 4.

20. The method according to claim 16 wherein the wave generator sends to the electrodes trains of pulses of various amplitude and duration.

21. The method according to claims 20 wherein the wave generator sends to the microelectrodes five trains of 25 pulses, 1 ms duration, repeated at a time interval of 500 ms.

22. The method according to claims 20 wherein a trains of triangular voltages consisting of 10 pulses are applied to the electrodes the interval between one train and another being 5 s.

23. The method according to claims 16 comprising substantially the following steps:

cultivating cells since the adhering stage is reached;

adding in the culture medium at least one compound to be electroporated in at least one single cell of the said cells;

selecting at least one single cell and at least one microelectrode on which said selected single cell is adherent;

generating at least one electric signal capable to electroporate said at least one single cell with said at least one compound to be electroporated and driving said electric signal to said one microelectrode on which said selected single cell is adherent.

26. The method according to claim 23 wherein the compounds to be electroporated are drugs, genetic constructs and proteins.

4. A biochip comprising an array of individually driven microelectrodes comprised on a suitable insulating layer mounted on a solid substrate;

means to electrically connect said microelectrodes to a switching system;

a cell culture chamber where the cells can be grown and adhere in contact with said array of microelectrodes on a surface formed by said insulating layer containing said array of microelectrodes on said solid substrate.

5. The biochip according to claim 4 comprising a semiconductor substrate as the solid substrate covered with an insulating layer comprising said array of individually driven microelectrodes of a size comparable to the cell to be electroporated, and mounting a cell culture chamber with an opening mounted, in turn, on a support made of dielectric material, said microelectrodes being electrically connected via conductive traces to conductive pads electrically connected, in turn, to a couple of external parallel connectors through wire bonding covered by an outer portion of the cell culture chamber encircling the opening, being said cell culture chamber with the opening mounted over the top of said semiconductor substrate covered with the insulating layer both attached on the dielectric support.

6. A biochip, according to claim 5 comprising two further electrodes integrated in the semiconductor substrate covered with the insulating layer and acting as a ground reference.

7. A biochip according to claim 5 wherein the semiconductor substrate covered with the insulating layer is a silicon substrate covered with an insulating layer preferentially of SiO₂.

8. The biochip according to claim 5 wherein the solid substrates is transparent.

9. The biochip according to claim 5 wherein the dielectric support is vetronite, glass or ceramic.

10. The biochip according to claim 5 wherein the microelectrodes of the array have a size with a surface of at least ten percent of the total cell membrane and preferably a diameter ranging from 1 μ

m to 50 μ

m.

11. The biochip according to claims 4 wherein the microelectrodes are of conductive or capacitive type.

12. The microelectrodes according to claim 11 consisting of conductive microelectrodes obtained over a silicon substrate covered with an insulating layer preferentially of SiO₂, said microelectrodes having connecting traces wherein said microelectrodes and their connecting traces being made by a “

sandwich”

of two titanium nitride, TiN, layers and an aluminium layer, covered with a gold layer on their active surface.

13. The microelectrodes according to claim 11 wherein said microelectrodes are realized using Metal Oxide Semiconductor, MOS, technology.

14. The microelectrodes according to claim 13 consisting of a silicon p-type substrate in which two n-doped regions, a drain and a source, are implanted with conventional microelectronic techniques, a gate of these electrodes being realized in n+ doped polysilicon and is common to all devices in a row, word line, the drain of all devices in a column being connected together by using a metal contact plug and a metal line, the source of the transistor being connected via a metal, usually tungsten, plug to a gold layer which acts as the active electrode.

15. The microelectrodes according to claim 11 consisting of a capacitive microelectrode obtained with an insulating substrate, a metal and a thin insulating layer said microelectrodes being separated by insulating material and covered in non exposed areas by a passivation layer.

19. The method according to claim 16 wherein said biochip comprises microelectrodes according to claims 11.

24. (canceled)

25. (canceled)