This invention relates to an apparatus for and method of introducing a substance into an object.
More particularly, but not exclusively, the invention is capable of introducing substances into small objects, such as for example, cellular material or cells. The substance introduced may be a transfecting agent such as: a chemical, molecule, protein, virus, prion or DNA material.
Previously material has been introduced into cells by way of a syringe-like device. These syringe-like devices have to be operated by hand or by complex robotic systems, and in consequence the injection process has been very slow.
The present invention arose to overcome this and other problems associated with syringe-like devices.
It is an object of the invention to increase the efficiency of injection of material into small objects such as cells by the presently-known process of electroporation. In this process cells suspended in a medium are exposed to an electric field sufficiently high to cause the membrane to become permeable. In the present art, cells are suspended in a medium containing the species required to be injected. When the membrane has been made permeable, the species to be injected will either diffuse or be driven by electrophoresis into the cell. After a time the cell membrane will recover from the effects of the pulse and become non-permeable once more. In the present art high concentrations of cells are used, together with macroscopic (ie, with typical minimum dimension of order 100 μm) electrodes. This means that in general, more than one cell will be in series with each of the field lines in the system—the precise number being random. Hence the field experienced by the cells in the system can very greatly, from below that required for membrane opening, to above that which the cells can stand and remain viable. It is an object of the invention to provide a microfabricated system, with characteristic dimensions considerably smaller than those encountered in presently used electroporation devices, where cells can be electroporated and material injected in conditions which are very similar from cell to cell, allowing uncertainties of field strength and timing to be reduced.
Microfabricated devices for handling cells and exposing them to electric fields are known. Ayliffe et al (IEEE J MEMS 8(1)50-57 (1999)) show microfabricated channels with micro-electrodes which can be used for electrical impedance measurements on liquids and cells, the latter aimed at detecting the type of cell present and measuring (if possible) some of its properties. The device is not designed for the purpose of electroporation and no mention is made of this. Tanaka et al (U.S. Pat. No. 4,894,343) disclose a microfabricated device for handling cells in wells, designed for the purpose of fusing together two cells located in the same well. Their device comprises an array of wells etched in silicon which are designed to trap the cells, while allowing liquid to move past them through outlets in the bottom of the well. However, the device is not designed to be optimal for electroporation; the present invention uses an improved design.
According to the present invention there is provided an apparatus for introducing a substance into an object comprising: means for introducing the substance to the object; and means for causing permeability of the wall of the object so as to permit said substance to enter said object; characterised in that the means for causing a permeability includes at least one electrode, dimensioned and arranged to form the permeability in the wall of the object upon application of a voltage pulse.
Preferably a characteristic dimension of a channel through, or along, which an object passes or flows is of the order of 50 μm, more preferably it is less than 30 μm and most preferably less than 4 times the diameter of the object.
Objects or cells suspended in a liquid medium are introduced into a chamber in which the apparatus(es) is/are located by way of a pump or gravity feed or other suitable fluid displacement mechanism, for example by electro-osmosis.
Preferably at least two electrodes are provided so that the object is located with respect to the electrodes so that a potential difference may be applied in order to render the object wall temporarily permeable.
Means can be provided to restrain or locate the object so that it is positioned with respect to the or each electrode. An advantage of locating the object is that it is positioned in a particular part of a predetermined electric field. Consequently the electric field may be applied with greater precision.
A proximity detector is advantageously included, so that when an object is in the correct location to be in a predetermined part of the electric field, the voltage pulse is applied automatically. Processing means, including electronic logic, may be used to improve and enhance this process.
Preferably there is provided a plurality of the aforementioned apparatuses arranged in an array. An advantage of such an array is that many objects may be acted upon in parallel. This increases throughput.
An array of apparatuses may be formed on a semiconductor substrate, such as for example, silicon or germanium. Proximity detectors, electrodes and processing means may be included on the substrate, for example, in a different layer of an integrated semiconductor structure.
In a particularly preferred embodiment DNA is introduced into living cells by rendering permeable the cell wall by the process of electroporation. DNA then enters the cell from the surrounding medium. Cells are supported in a fluid which is under sufficient pressure to cause cells to move with respect to the electrodes. Means for locating each object with respect to an electrode may comprise a mechanical or electrical structure. An example may be a well or well-like structure, formed for example by back etching a silicon substrate in which the cell locates. A mesh or sieve-like arrangement can be placed at the exit of the well so as to permit passage of fluid but prevent the cell from leaving the well. Preferably a pressure differential established across the substrate urges cells into the well-like structures.
As more cells are located in wells the pressure differential increases because less wells are available, through which fluid may flow. This increased pressure tends to force cells into the wells as they deform relatively easily. One way of preventing this from occurring is to obtain an indication of wells which are occupied and use this information to reduce or increase the pressure differential. This information is readily obtainable as the presence of a cell is known from proximity detectors and a counter in a microprocessor may be used to increment each time a well becomes occupied.
Preferably the apparatus is microfabricated from a biocompatible material. The microfabricated apparatus may include one or more microfabricated channels. These may be formed for example by etching in silicon. Wells or sites for introduction of material may be at a locus in a fluid flow channel.
The channel is preferably narrow, for example, between 1 and 5 times the diameter of the cells (which may typically be around 5-20 μm) to be electroporated. Such narrow channels are advantageous in electroporation, as a greater proportion of the electroporation voltage may be applied across the cell per se, rather than across the cell, any neighbouring cells and the supporting fluid. This enables the field experienced by the cell to be controlled precisely. In an alternative embodiment the channel may even be narrower than the diameter of the cell in its relaxed state. In this embodiment cells deform and flow along the channel and are in closer contact with the walls.
Alternatively the channel or well may be relatively wide except for a constriction in the region at which introduction of material occurs, the constriction, and/or eletroporation electrodes may be designed so that pores, opened in the cell membrane to allow introduction of material, are preferentially oriented at a source of the material.
In a microfabricated device electronic logic may be used to control the amplitude of the electroporation voltage pulse or sequence. The logic circuitry may be integrated within a semiconducting substrate, for example using CMOS, DMOS or bi-polar components, fabricated in a convenient process sequence. Preferably the substrate also forms a support for microfabricated channels. Post-processing techniques can be used during manufacture of the substrate to interconnect electronic components to electrodes in the flow channel(s).
Integration of a moderately high-voltage (typically 5-25 volts although other voltages may be used) switch device (used to control the electroporation voltage) is especially advantageous as the minimal electrical impedance between the switch and the channel may be used to enhance control of the electroporation parameters. The inclusion of one or more capacitive elements adjacent the switch is most preferable as this enhances the ability to source current rapidly, without requiring complex current distribution circuitry to the switch. This may be of particular advantage when there is integration of multiple electroporation devices for example in an array. It is an advantage of the present device that as the channels are small, the electrodes can be made close and the voltages needed to achieve electroporation are low and easily controlled.
The introduction of electronic components or logic circuitry, by an active substrate technique, is elegant and is of especial benefit when an array or arrays of electroporation devices are co-fabricated on a common substrate. The possibility exists to substitute or augment such components or circuitry by attaching additional microelectronic components, at appropriate positions, to the substrate. Such components may be attached by surface mount, die and wire bonding, TAB bonding or flip chip bonding. The attachment of devices using conductive adhesive means is especially preferred since this minimises any thermal stresses imparted to the structure during fabrication.
Preferred devices and attachment means and capacitor devices are attached by surface mount (including attachment by conductive adhesives) or by wire bonding. The aforementioned devices are particularly preferred where the substrate is passive or contains low voltage components. Analogue processing circuitry, analogue-to-digital converters, digital signal processing devices, microcomputing or microcontroller elements, and communications devices may also be integrated onto the substrate. The latter devices include optical communication devices. Integration facilitates connection of processing or control circuitry to external processors, such as a microprocessor for closed loop flow control and/or electroporation pulse application.
Preferably sensing means is provided which operates in conjunction with an electrode and a common ground, or a pair of electrodes, to interrogate a channel or well for determining the presence of a cell.
Preferably control means controls the instant of electroporation pulse timing in collaboration with the detection of a cell in the well or channel. For example, a microcontroller, timer or state-machine may be integrated and used to control the instant of application and/or amplitude of an electroporation pulse, in response to a signal indicating the presence of a cell.
Preferably means is provided to determine the state or condition of cells following transfection, indicating whether a cell is unaffected, has had material introduced successfully, or is damaged. For example apparatus as described in published International Patent Application No WO-A-9402846 (BTG) may be utilised for this purpose. Thus it is possible to characterise the cell, at a locus or loci both prior to and following an attempt at introduction of material, and to determine the success or otherwise by the difference in the cell's response rather than by an absolute calibration.
Preferably processing means responds to an external indication of the presence or state of a cell in the electrode vicinity. A trigger may be provided by an operator or automatically by a digital i/o card in a common microcomputer. The trigger may in turn be derived by image processing means such as a video microscope image of the channel.
Detection or monitoring of cells may be done by optical means. Cells may have a fluorescent component introduced into them when material is introduced. This allows automatic fluorescence detection of treated cells, for example by using a video microscope and/or other image processing means, to detect successful introduction. This data may be used to signal the presence of the cell in the electroporation apparatus.
Integrated components or circuitry and an associated well or channel, in the or each electroporation apparatus, are provided with a unique address and a communication means is provided allowing communication to and from a microprocessor. Preferably communication is via a common link or bus.
In the case that the support substrate is silicon the possibility exists to view the cell handling structures through the silicon using suitable infra red radiation. In such an embodiment care must be taken in the layout of the structures to prevent obscuration of the radiation path. An advantage of this embodiment is that the device need not use any member which is optically transmissive in the normal visible band, but is infra red transparent. As infra red radiation may inhibit any fluorescence detection, any infra red detection should be performed at a separate location to fluorescence detection.
Subsequent to the introduction of the material into the cells, there is preferably provided sorting means to direct cells into a collection flow or to a waste flow depending on the result of the introduction. Such sorting may be achieved by a number of methods and include: electrophoresis, dielectrophoresis, “optical tweezers”, and/or directed pressure pulses.
Means may be provided to destroy cells which have not been treated in the desired manner. Such destruction may be achieved by killing the cell(s) while leaving it then essentially physically intact or by physically disrupting the cell(s). Preferred means of achieving this include: electrical disruption of the cell, essentially by overly vigorous electroporation, the introduction of a cell lysing agent; or rapid local heating of the cell or fluid medium in the vicinity of the cell. A micro-heating element or directed, pulsed infra red radiator may be used for this purpose.
In cases where the electroporation apparatus has electrical connections routed about or around it, for example in a highly integrated active substrate, it may be desirable to provide electrical guard bands suitably disposed around portions of the fluid handling structure so that any electric field applied to the fluid is reduced sufficiently so that there is minimal deleterious effect on cells flowing in the channel.
Optical components, such as waveguide optics, may be integrated in processed layers of the substrate which are preferably fabricated in similar processes to those defining fluid channels. Such optical components may include waveguides for interrogation of the cell or support medium in the fluid channel. These may include evanescent field coupling Alternatively optical components communicate to external signal processing means. Optical components include structures interfacing with fibre optic elements such as etched silicon V-grooves.
Arrays of apparatuses may utilise common external connections for supply of fluids, cells and power supplies and may be imaged in parallel using suitable video microscopy means.