Electroporation cell with arc prevention/reduction

Details Electroporation cell with arc prevention/reduction Electroporation cell with arc prevention/reduction

1999-10-05

2001-07-10

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Apparatus

Mutation or genetic engineering apparatus

C435S461000, C218S004000, C361S006000

Reexamination Certificate

active

06258592

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates generally to cell transfection, and, more particularly, to an apparatus and method of electroporation that prevents and/or reduces arcing across a cuvette containing a sample of biological cells.
Electroporation is a process by which high-voltage (typically high energy) electric potentials are used to create temporary holes (“pores”) in the walls of biological cells. These pores allow the passage of large molecules (e.g., DNA) into the cell, before the cell eventually closes the pores. As a consequence, electroporation can be used to program a cell to produce proteins specified by the DNA (bacterial cells for example can be caused to produce human insulin). Electroporation is, therefore, an extremely powerful tool, since a 5-msec application of a high-energy pulse can create openings in millions of cells at the same time. Cells that have received DNA in this manner can then be grown in a nutritive broth to produce an aggregate, which generates large quantities of some desirable complex molecule. Typically, a biological cell can produce compounds in seconds or minutes whereas a conventional synthesis may require a week or more when performed in a laboratory using chemical synthesis procedures.
Previous patents (U.S. Pat. Nos. 4,750,100, 5,656,926 and 5,642,035), each of which is incorporated herein by reference, describe the use of semiconductor-controlled rectifiers (SCRs) in a stacked-cell arrangement to produce a solid-state high-voltage (HV) switch capable of controlling 3000V pulses at currents of 1500 amperes (or higher, depending on the SCR). Increasing the number of SCR cells allows the control of higher voltage pulses. The general method of producing such pulses is shown in
FIG. 1
attached hereto.
In
FIG. 1
, a charging means
4
, consisting of a linear or switcher current source, is controlled by a microcontroller
10
through an isolated control line
18
. The microcontroller
10
turns on the charging means
4
, thereby causing charge to accumulate in an HV capacitor
6
. As the voltage across the HV capacitor
6
rises, the microcontroller
10
monitors the increasing voltage, using a voltage divider
12
consisting of two or more resistors. When the voltage has reached a predetermined value, either by embedded program or external setting by an operator, the microcontroller
10
triggers the HV switch
8
(consisting of the SCR cells) through a trigger circuit
14
. The HV switch
8
effectively connects the HV capacitor
6
to the output of the system, which in turn connects to a cuvette
16
or other sample holder containing cells and DNA (or other) compounds. The resistance of the sample in the cuvette
16
may be effectively 10 ohms at high voltage. Hence, without a current limiting resistor, large currents could flow and destroy the HV switch
8
. Later embodiments, such as those in U.S. Pat. Nos. 5,656,926 and 5,642,035, for example, use a 1.5-ohm series resistor with feedback through the microcontroller to compensate for voltage drops across the resistor.
Another concern in the use of such a system is arc-over. If the voltage at the cuvette
16
is too high, an arc-over may occur. Arc-overs lower the cuvette's
16
effective resistance to 1.0 ohms or less. Hence, at 3000V (with 1.5 ohms current limiting resistance), 2000 amperes may flow. Typical SCRs used for such an application will tolerate such currents. However, the arc will cause a visible and auditory event at the cuvette
16
, which may expel the sample, destroy cells, and startle the operator.
In the electroporator system of U.S. Pat. No. 4,750,100, no specific current limiting resistor is incorporated, since such a resistor would cause a large voltage drop. (The resistance required by the resistor would have to be 1.5 ohms, which is significant compared to that of the sample, whose resistance is 10 ohms.) U.S. Pat. No. 4,750,100 does, however, describe the use of the SCR cells to produce an appropriate HV switch. In U.S. Pat. Nos. 5,656,926 and 5,642,035 a system that measures sample resistance is included. This allows the microprocessor to compensate for the voltage drop across a current limiting resistor by charging the HV capacitor to a somewhat higher voltage. Other novel portions of the design are described in the same two patents.
Lack of arc protection in the electroporator system of U.S. Pat. No. 4,750,100 was acceptable since arcs were infrequent, until the system was later used on bacterial cells. Bacteria require the use of higher voltages, thereby increasing the likelihood of arcs. Bacteria electroporation in those systems requires a special box containing the current limiting resistor (which might be forgotten by the user to the detriment of the instrument) or the incorporated arc protection of the instruments disclosed in U.S. Pat. Nos. 5,656,926 and 5,642,035.
A better system would be one that, not only provides protection from arcs, but one that limits the time in which an arc is applied to a sample. Such a system would have less of a chance of startling users and would prevent destruction of valuable cells. The present invention provides a solution that addresses these issues.
SUMMARY OF THE INVENTION
The present invention provides both a system and method for reducing or quenching arcs during electroporation.
Generally, the system and method of the present invention provide for a current diverting circuit, which is operable to divert current away from a sample of biological cells involved in an electroporation session, so that the cells are not damaged by excess current should arc-over commence.
According to one aspect of the invention, the salient components of the system include: a charge control circuit; a high voltage capacitor, which accumulates charge provided by the charge control circuit; a microcontroller, which is capable of controlling the transfer of energy to a cuvette containing a suspension of biological cells; and a current diverter.
According to another aspect of the invention, the microcontroller monitors the voltage drop across the high voltage capacitor by employing a voltage divider at one of its inputs. When a first predetermined voltage is reached, the microcontroller takes action to cause the charging means to decouple from the high-voltage capacitor and trigger a first high-voltage switch, which functions to couple the high voltage capacitor to the cuvette containing the sample.
According to another aspect of the invention, the current diverter circuit includes a sense resistor, which is connected between the first high-voltage switch and ground. When the voltage drop across the sense resistor exceeds a second predetermined voltage, a current diverter switch is triggered, which functions to divert current away from the sample and then to ground.
In yet another aspect of the invention, a method is described which, when applied, reduces the amount of time during which a high voltage is applied to a cuvette containing a sample of cells when an arc-over event occurs or when the sample resistance is detected to be below a first predetermined value. The method comprises the steps of: (i) charging a high-voltage capacitor to a first predetermined voltage; (ii) triggering a high-voltage switch to couple substantially all of the first predetermined voltage of the capacitor to electrodes of the cuvette; (iii) monitoring the voltage applied across the cuvette; and (iv) triggering a current diverter switch when the monitored voltage is larger than a second predetermined voltage.


REFERENCES:
patent: 4923814 (1990-05-01), Marshall
patent: 5656926 (1997-08-01), Ragsdale

Affiliated with

Also associated with

Rating

Electroporation cell with arc prevention/reduction does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Electroporation cell with arc prevention/reduction, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Electroporation cell with arc prevention/reduction will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2435198