BRIEF DESCRIPTION OF FIGURES
FIGS. 1A-D illustrates a typical embodiments of the device for CDEB perforation of this invention;
FIGS. 2A-B illustrates a typical embodiment of the current-voltage control of the device for CDEB perforation of this invention;
FIGS. 3A-G shows microscopic images of micro holes formed in polypropylene and glass substrates and corresponding current-voltage curves as well as a typical time course of current and voltage during processing;
FIG. 4 illustrates an embodiment of a device used for electrophysiological measurements with biological (lipid) membranes using the carrier device of this invention;
FIG. 5 illustrates an embodiment of a device used for electrophysiological measurements with biological cells using the carrier device of this invention.
FIG. 6 shows a time course of the trans-carrier current during seal formation of a biological cell and recorded single ion channel currents after seal formation.
DETAILED DESCRIPTION OF THE INVENTION
The device and methods of this invention can be used for the formation of hole and channel like structures in insulating substrates, in particular useful for electrophysiological and other measurements and set-ups where independent access to parts of biological membranes and cells is required. The terms “carrier” and “substrate” will be used synonymously and interchangeably throughout this patent application, with the term substrate referring more to the actual material to be micromachined and the term carrier indicating its actual function.
The formation of high aspect ratio hole (i.e. ‘tunnel’ or ‘channel’ like) structures in insulating or semiconducting substrates with current micromachining tools such as reactive ion etching or laser ablation is difficult, expensive and in most cases limited by size and geometry. However, for hole structures in insulating carriers used for the independent access of membrane parts, as e.g. patch clamp on a chip or BLM measurements, the precise location of the hole structure is less important compared to e.g. microelectronic circuits. Also, the hole diameter can vary within a rather large range (e.g. up to 50%) for the intended biological applications without significantly impacting the experimental quality and results. The possibility to form the hole or tunnel like structure at a largely arbitrary position at the substrate/carrier area reserved for membrane/cell access with only a roughly defined diameter provides the basis for the application of micro machining techniques that have lower precision than standard micromachining techniques. However, the generation of high aspect ratio holes in substrates not amenable to micromachining techniques, such as glass or quartz, have remained a formidable engineering challenge until the advent of the present invention.
A physical phenomenon that can be used to form small holes with high aspect ratio, but lacks otherwise high precision required for e.g. microelectronics, is “dielectric breakdown” (DEB). This phenomenon occurs in insulators in electric fields (e.g. insulators sandwiched between two electrodes) when the applied voltage and electric field strength, respectively, increases to values where an “insulator-to-conductor” transition occurs. Due to Ohm's law I=V/R (I . . . current, V . . . voltage and R . . . resistance), a sudden increase in current, and consequently power dissipation P=R×I2, between the electrodes and through the insulator is caused by a significant reduction in electrical resistance. Along the current path insulating material is transformed or removed (e.g. by burning, evaporation or material ejection) which can lead to the appearance of cavities, hole or tunnel like structures. This phenomenon is known for decades and mostly a parasitic effect in high voltage circuits or sensitive electronic components as e.g. FET transistors (gate electrodes). It has also been used in industrial environments to e.g. perforate thin plastic packaging sheets to permit gas exchange. Because it appears difficult to separate between the various effects high voltages of different frequencies exert on dielectric materials, in this patent application the term DEB is used for all voltage induced electric/dielectric processes leading to a local (if locally applied) change in the material structure of the substrate. In particular, this concerns local increases in substrate temperature upon voltage application, which can be used to visibly modify the substrate material. For the primary applications in electrophysiology, these modifications will be hole formations.
DEB has been used in the past for the formation of small holes (ca. 20-50 um minimum) in plastic substrates for BLM measurements. However, due to the employed DEB devices and methods, which did not allow for a precise power control (i.e. control of voltage, current magnitude and duration) during and after DEB, micro holes with reproducible diameters below 20 um were not achieved. But micro holes significantly below this diameter are required for carriers for patch clamp like measurements (cell size usually<25 um) and stable and commercially usable lipid membrane (Note: the BLM stability is inversely correlated to the membrane diameter) devices. Until now, it is not known that DEB has been used before in a precisely defined and controlled manner for reproducible micro-structuring of insulating substrates intended to carry small (i.e. less than ca. 10-15 um) biological membranes or objects thereof at the micro-hole site.
To manufacture holes and other structures within a defined range of spatial dimensions by DEB, the energy dissipation during and after DEB, according to the present invention, must be accurately controlled (controlled DEB or CDEB). Because the dissipated energy is the product of current times voltage times duration, all three factors are controlled. FIG. 1A shows a possible realisation, in which the voltage is controlled by a process controlled and optionally current limiting high voltage power supply. Depending on the properties/control characteristics of the voltage source, the current may also be limited by an optional resistor R, which is in series with the substrate. This has the advantage that for a not sufficiently fast power supply control or parasitic capacitances which may render the control of the power supply not fast or precise enough, the maximum current during CDEB can not exceed I=V/R, with V indicating the amplitude of the applied voltage. The CDEB duration and consequently voltage application is e.g. set by a timer which is triggered at a preset trans-substrate current level usually indicating the onset of the DEB process. The onset of the DEB process is indicated by a very steep and strong current increase. Because of the exponential nature of this current increase during DEB, CDEB requires a fast trigger. In some embodiments it is sufficient to use a somewhat slower trigger control/power supply control in conjunction with a current limiting resistor R (see above) because the exponential current rise is limited by the resistor to a preliminary value of I=V/R limiting the power dissipation and consequently tending to increase the CDEB interval. However, this method works preferably for polymers and similar materials. For all presented data in this application, the trigger speed and consequently time control precision of the CDEB process was in the range of 0.1-1 ms for non-critical CDEB processes (i.e. low DEB energies) and 1 nsec-100 usec for high DEB energies. A possible realisation of a suitable high voltage source is illustrated in FIG. 2. Low DEB energies refer to maximum trans-substrate currents usually in the range and below 10−5 A.
FIG. 3A shows a micro hole formed with CDEB in polypropylene (upper panel) as well as the current-voltage trace recorded when the trans-substrate voltage was raised to the critical DEB value (lower panel). Smaller holes (diameter<1 um) were consistently produced by further limiting the current upon an increase in the series resistance R.
The distance between the electrodes and carrier to be structured can be varied. If the electrodes touch the substrate (‘contact mode’), the necessary DEB voltage is reduced to a minimum. However, contaminations and mechanical influences on the substrate deriving from the electrodes may occur. Using a gap between the substrate material and the electrodes may increase the necessary DEB voltage, reduces however the risk of electrode interferences with the substrate surface.
A gap between substrate surface and electrode allows for the ionization of the gas molecules between them, providing the means for a modification of the substrate surface through activated gas molecules. For this the gas composition between the electrodes and substrate is controlled in such a way that during DEB the ionized gas molecules interact with the substrate surface in a manner beneficial for the intended application (e.g. cell adhesion). An example is the usage of pure oxygen which leads to the generation of activated oxygen molecules/ions/radicals during DEB which in turn can oxidize the substrate surface. Another way to concurrently modify the surface during DEB is the prior coverage of the surface with materials that, upon the ionization and heating process during DEB, undergo a chemical modification beneficial for the application of the substrate (e.g. for better membrane adhesion). The surface properties of the CDEB formed hole and its surroundings can also be controlled by selection of a substrate material that during DEB is fully or in part transformed into a material of choice.
The electrodes can be surrounded by an insulating material such as PDMS (polydimethyl siloxane) that also tightly seals to the substrate surface. This avoids DEB process bypassing the substrate and going through the adjacent medium (e.g. air) and consequently allows to structure also substrates with small total surface areas. Another possibility of avoiding DEB processes bypassing the substrate is the usage of substrates surrounded by media that have a much higher breakdown voltage than the substrate material itself (e.g. silicon oil). The electrodes may also be surrounded by liquids of various dielectric properties (e.g. water, dichloromethane) to modify the DEB outcome as well as to modify the temperature distribution at the structuring site.
The classical DEB method as well as the here described CDEB method still lack the ability to perforate most substrates of interest, e.g. substrates that do not easily melt/burn, have crystalline structures, are to brittle or require DEB voltages that are not useable for substrates in practice (glass, quartz). In order to make CDEB accessible to such materials, the necessary DEB voltage must be decreased or at least modulated and, in some cases, material properties must be changed or initialized before the actual DEB hole production process takes place so that the material is e.g. soft enough as not to break during hole production (e.g. glass). In theory, CDEB structuring can be applied to essentially any insulating material, since all insulators show at some specific electric field strength a full or partial transition into a conducting state. Consequently, a wide selection of substrate materials ought to exist allowing for an optimal selection of substrate/carrier parameters such as membrane and cell adhesion and electric/dielectric properties for e.g. Patch Clamp on a Chip applications.
To extend the CDEB method in general to become applicable in practice to many substrate materials of interest in biology, physics and technology, the reduction of the insulator-to-conductor transition field strength by raising the substrate temperature is a central part of this invention. Heating the substrate, either extrinsically with an additional external energy source or intrinsically by e.g. dielectric losses caused by AC voltage components before CDEB takes place, can both sufficiently reduce the required DEB voltage and alter the material properties so that materials, where the dielectric breakdown point is usually difficult to achieve or side effects come into play, can be microstructured. To additionally better define the CDEB process location at the substrate, heating can be locally restricted. Heating the substrate or usually parts thereof in a defined manner makes materials accessible to CDEB that usually can not be modified at a useable thickness or that tend to break because of brittleness at normal (ambient) temperatures. For instance, making round holes in glass cover slides (e.g. Menzel S1) by ‘normal’ CDEB is virtually impossible due to the required high voltages and also the breakage of the glass slides once DEB takes place at these high voltages; injecting an appropriate amount of additional heat at the intended CDEB site reduces the electric resistance sufficiently to initiate DEB leading to round and largely smooth holes in the slide. An intended effect of this method is the production of high aspect ratio holes. Because the necessary DEB voltage is lowered by heat injection, relatively thick substrates (compared to e.g. reactive ion etching) can be structured. Aspect ratios larger 100 in materials such as glass (e.g. Menzel S1) are possible and have been achieved according to the present invention. It is also clear that not only heat but essentially any kind of energy lowering the voltage at which the “insulator-to-conductor” transition (DEB) takes place can be applied to support the CDEB process. In particular ionizing radiation also promises to lower the DEB voltage. The invention considers and claims the use of high energy radiation in order to lower the electrical resistance of the substrate material and to consequently induce DEB at practical voltages. Examples are x-rays and ion beams.
Lowering the voltage required for DEB also reduces the rate with which electrical energy deriving from the applied voltage (source) is transformed into heat during DEB (P=V2/R, where R is mainly the resistance of the substrate, which is likely to drop even faster with increased voltage due to faster heating during DEB). This moderation of energy injection into the substrate is important to e.g. avoid excessive thermal expansion of regions of the substrate or melting/burning of organic substrates. In particular for brittle materials such as glass this may contribute in avoiding an unspecific breaking of the substrate.
The invention claims the particular use of this thermally supported DEB process. For materials difficult to perforate by DEB under ambient temperature conditions, such as e.g. glass and quartz, a heat source is added to the perforation device (FIGS. 1B, 1C, 1D). We refer to this method as extrinsic heat supported CDEB. Heating the substrate can achieve the following: (I) softening (if necessary up to the point of melting) of the substrate material or parts thereof (II) reduction of the necessary DEB voltage caused by a lowered electrical resistance of the substrate.
The heat and energy source, respectively, can inject energy/heat in different ways. It is possible to apply energy/heat from one or both sides (referring to the position of the electrodes) of a substrate. Various heat sources are suitable, e.g. lasers (FIG. 1C, e.g. infrared laser for glass), heating filaments (FIG. 1B) and flames. Due to the fact that flames consist of (partially) ionized gas molecules and consequently have a higher electrical conductivity than cold gas (e.g. surrounding air) they can be used as an electrode for the voltage application during CDEB (FIG. 1D). For this reason a metal or other electrically conducting part which is in contact with the flame (e.g. the metal opening of the burner releasing the flame) is connected to the DEB voltage source.
The invention claims the use of directed and locally restricted heating of the substrate with the goal to induce only locally the above described heating effects on the substrate material and consequently direct the location of the CDEB process on the substrate. As an example, the flame of a gas burner is focussed and positioned at the substrate surface where the hole is to be formed (FIG. 1D). Similarly a laser spot can be positioned at the substrate surface (FIG. 1C). The combination of high precision laser spot positioning and normal CDEB defines a device and method for high precision CDEB micro-perforation.
The invention claims that (locally) adjusting the substrate temperature to specific levels or ranges is a way of controlling the hole/channel properties. This becomes immediately clear considering e.g. the differences in viscosity, surface tension and electrical resistance of the substrate material at different temperatures. Also the control of the heat distribution across the carrier is an additional method to modulate the CDEB outcome on the hole/channel properties. Appropriate ways of controlling the heat distribution involve the size and placement of the heat source (e.g. heating filament size and distance to the substrate surface), the amount heat coupling from the heat source into the substrate per time unit as well as the duration and possibly modulation of the heat application. In tests it was observed that changing the distance between the heating filament (1×1 mm2 active area, FIG. 1B) and the substrate surface (Menzel S1) by only a few micrometer (total distance filament—substrate surface ca. 100 um) would significantly shift the amount and distribution of the heat igniting the actual DEB process (voltage set prior to heat application to ca. 8-20 kV) and consequently the outcome of the entire DEB process, ranging (with all other parameters fixed) from no holes in the substrate to perfectly round holes. At short distances (ca. 25-80 um), holes appeared mostly round (FIG. 3C). At distances of significantly more than 100 um, the Pt heating filament had to be excessively heated for DEB initiation so that metal depositions appeared at the substrate surface. In most cases, metal depositions are not wanted and this distance can be used to define the largest useful distance. Of course, if for certain applications metal depositions are wanted, the deposition process can be combined with the CDEB process. Furthermore, the substrate may be ‘clamped’ at some locations to certain constant heat reservoirs to maintain the desired heat distribution.
An additional function can be assigned to substrate heating and is part of this invention, which may occur either by an extrinsic heat source or an appropriate trans-substrate/trans-hole current generated after CDEB. Applying the heat beyond the dielectric breakdown, the produced structures can be post-processed by melting/annealing/tempering. This is an appropriate way to e.g. change the diameter of CDEB produced holes, to smoothen the surface roughness inside and outside the hole mouth or to eliminate mechanic tensions of the material surrounding the hole. In tests, the hole diameter could be reduced up to a factor 1.6 (as determined by conductance measurements in saline solution) by such a prolonged heat application.
Substrate heating and CDEB can be combined in various ways to achieve the desired holes/channels and surface properties. The invention uses most commonly: (I) heating of the substrate to a preset value and consequent application of the DEB voltage and (II) application of a specific DEB voltage and heating of the substrate until DEB occurs. In both cases, heat and voltage may be reduced after DEB with or without a delay in a way suited for the CDEB process, e.g. abrupt reduction or ‘fading’ out. In fact, by controlling voltage and current after the actual CDEB, the formed structures may be post-processed. For instance, the heat produced by the electric arc passing the substrate at a hole site modifies the hole by melting surface material. That way, the structure itself as well as its surface properties can be modified.
A very simple and elegant way of combining substrate heating source and DEB source is the use of a single modulated or alternating voltage source. We refer to this method as intrinsic heat supported CDEB. In this case, a method consists of at least two components: (1) local heating of the substrate through (1A) dielectric losses of the substrate material induced by a changing voltage/electrical field across this substrate region and/or (1B) through electric arcs forming between the electrodes and substrate surface and (2) DC voltage induced normal CDEB. A suitable device employing this method can consist of only two electrodes, which are connected to a controlled voltage source providing the necessary AC-DC voltage superposition as well as any kind of a sufficiently insulating mechanic support for the substrate (FIGS. 1A and 2). For standard microscopic cover slides (Menzel S1 18×18), DC voltages of 0-15 kV and AC voltages of 10-40 kV and 4-100 kHz were commonly used to prepare micrometer sized holes (usually 1-7 um). The electrode distances (e.g. a pointed Pt-wire 0.5-2 mm in diameter) were usually 0.5-3 mm. Other parameters (e.g. higher frequencies) work as well but were outside the voltage supply limits. The dielectric losses and consequently heating of the substrate material are determined by factors such as duration, frequency and duty cycle of the AC component of the applied voltage, voltage amplitude and dielectric material properties. Even in the absence of dielectric losses, the invention claims the use of electric arcs forming at sufficiently high AC voltage amplitudes and frequencies capable of heating localized substrate areas sufficient for DC voltage supported DEB hole formation. Without wishing to be bound by any theory, the rational behind this method is that with increasing frequency, the impedance across the substrate drops due to an increase in capacitive currents Ic according to the equation: Ic=C*dV/dt, where C is the capacitance of the substrate-electrode assembly and dV/dt is the voltage change per time. Consequently, the voltage drop across the substrate is reduced with increasing frequency. For a given voltage amplitude this leads to a higher voltage drop outside the substrate. However, since the (usually) gas molecules outside can be much more easily ionized than the substrate material in most cases, the gas ionization at sufficient voltages leads to the formation of electric arcs between the substrate surfaces and the electrodes. These electric arcs are often hot enough to gradually melt and sometimes remove the substrate material they are touching.
It is crucial for all thermally supported CDEB processes and part of this invention, that substrate material at the structuring site is sufficiently softened or even molten before the actual hole producing CDEB step takes place. We refer to this step as heat initialization. In particular for brittle or crystal materials this becomes important. If this is not taken into account, the substrate may break because of brittleness (usually at the structuring site) and become unusable. However, even without proper heat initialization, the voltages can sometimes still be raised until DEB occurs, usually causing material to break out of the substrate, forming a brittle structure not useable for most applications. For instance, this can be observed when placing a thin glass slide between closely spaced high voltage electrodes (e.g. electrode distance<0.2 mm to substrate, substrate Menzel S0/S1 cover slide) and the voltage is raised to the point of DEB without any heat application (usually in the range of V ca. 30,000 V (DC)). Proper heat initialization, either by extrinsic or intrinsic heat sources, is therefore central to all described methods and devices when high voltages are applied that can also break the substrate material. This unwanted process breaking process is frequency dependent and has been observed to become more likely with decreasing frequency. It is important to notice that parameters for intrinsically heat supported CDEB can be chosen in such a way, as part of this invention, that heat initialization and the actual CDEB perforation step move seamlessly into each other, i.e. the onset of the CDEB process is chosen (controlled by preset parameters such as voltage amplitude and frequency and modulation) to serve as heat initialization. This allows for a much reduced effort in process control.
A special and very useful realisation of this invention is the usage of alternating or modulated voltages with no or only small DC components for CDEB. In such a case, an AC voltage is applied across the substrate region to perforate. Without wishing to be bound by any theory, the dielectric losses of the substrate upon this AC field/voltage application and/or the electric arc which may form lead to such a strong local substrate heating that a hole can form through the substrate (i.e. essentially pointing from one electrode to the other). The actual hole causing process usually occurs through thermally induced very sudden volume increase as well as evaporation of the substrate material, which in turn leads to an ‘ejection’ of liquid and gaseous substrate material out of the forming hole structure. As described before, in many cases parameters must be also controlled in such a way as to delay the hole producing step until the substrate material is soft enough. Only for this timing, it concerns in particular voltage amplitude and frequency/duty cycle. For standard Menzel S1 18×18 glass cover slides AC voltages of ca. 20,000 V, wire electrode distances of ca. 0.5-6 mm, and frequencies of 2-100 kHz were successfully tested for substrate perforation. Perforation times varied between ca. 10-600 ms. Other frequencies and voltage amplitudes are certain to work as well but were outside the limits of the perforation device/setup that was used. However, for most parameter sets it is important to tightly control the duration during which current flows through the substrate. In particular, after formation of the hole, which occurs in a sudden manner, the now increasing current must usually be immediately reduced or fully turned off to avoid a further temperature increase of the substrate surrounding the hole, which may lead to a closure of the hole by molten substrate material. In practice it is favourable to not only apply a constant AC voltage but to apply it intermittently, that trains of AC voltage intervals with adapted parameters (e.g. duty cycle and frequency that also may be adapted/changed during the pulse train) in order to control the perforation process so that no cracks and other unwanted structures form inside the substrate. For standard microscopic cover slides (Menzel S1), trains of 1-30 pulses of 10-400 ms duration separated by 0-200 ms off-time were commonly used to prepare micrometer sized holes. Typical AC voltages were between 10-20 kV with frequencies of 10-70 kHz and varying duty cycle (typ. 25-75%). The fast trigger (10̂−7 sec delay) for pulse termination was usually set at trans-substrate currents between 0.1 to 100 nA, preferentially 1-20 mA. These current values could not be directly measured but were estimated. The highly precise trigger control itself used experimentally determined and interpolated current values expressed in random units. Also a temporal variation of these parameters during the pulse train (e.g. the subsequent pulses of the train differ in AC power and frequency and trigger current) is possible and has been successfully applied. For example, reducing the AC frequency from 60 kHz to 20 kHz during the pulse train improved the hole quality. Also, pulse trains were applied repeatedly, with each train termination triggered by DEB onset (i.e. trigger current level reached), yielding very small (diameter<2 um) and open holes in e.g. Menzel S1 glass slides. With the same cover slides it was observed that only at ‘parameter islands’ open holes were produced. In between, holes were probably closed by tiny amounts of molten glass moving into the center of the hole, probably by surface tension. As stated before, also with this particular method CDEB parameters can be chosen such as to combine heat initialization and perforation step.
To avoid cracks in the substrate forming after hole formation all CDEB methods can be combined with an additional tempering step. After hole formation, large mechanical tensions can form inside the substrate (e.g. with glass) at the hole location. Moving the substrate temperature up, e.g. in the range of the substrate softening temperature, usually reduces these tensions rendering the substrates long term stable and widely applicable.
Increasing the frequency of the AC voltage component is a method to better define the location of the forming substrate hole. This becomes immediately apparent considering the fact that the capacitive current component of the current flowing between the perforation electrodes increases with increasing frequency while the ohmic current stays essentially unchanged so that the overall current, which is increasingly dominated by the capacitive component, follows the direct way between the electrodes, more and more unaltered by ohmic obstacles. Since the capacitive current does not necessarily follow the ohmic path (i.e. lowest ohmic resistance), already existing holes or cavities and other structural or material property inhomogeneities lead to lesser deviations of the current path and consequently to a more precise hole location with increasing frequency. This extends the method to multiple perforations of one substrates with holes closely spaced. For applications in micro fluidics, biotechnology etc, which may require e.g. arrays of closely spaced cavities (e.g. for reaction chambers), the usage of higher AC frequencies leads to a method suited for the production of e.g. high density arrays of micro cavities and other structures (e.g. surface channels) suited for applications in these fields. To produce surface channels and cavities for such applications (instead of holes), the CDEB process is either terminated before full opening of the hole or prolonged so that the hole is closed again with molten substrate material. Moving the substrate during CDEB leads to the formation of channels. For these latter purposes the intrinsically heat supported CDEB method is clearly preferred.
The combination of micro-structured carriers made by thermally supported CDEB with the means for electrophysiological measurements provides the basis for new and inexpensive devices monitoring electrical currents through biological membranes. Here the carrier separates two or more fluid compartments that are only connected through the CDEB produced hole. The biological membranes to be analysed are placed on one side of the carrier across the hole sealing it tightly. FIG. 4 illustrates the usage of a CDEB micro structured carrier as support for an artificial lipid membrane in a BLM set-up; the lipid membrane is usually provided by a giant unilamellar vesicle positioned at the hole opening. FIG. 5 illustrates the usage of a micro structured carrier, processed by thermally supported CDEB, as support for a patch clamp type set-up with biological cells. For such measurements it is required that membranes adhere tightly (forming so called ‘giga seals’) to the surface of the carrier thus avoiding leakage currents bypassing the biological membranes. Microscopy glass cover slides have shown to work well as basic carrier/substrate material. After tight membrane sealing, currents measured across the carrier (usually in voltage clamp mode) provide insights in membrane properties, in particular about the embedded ion channels and their control and interaction with e.g. other molecules and the applied voltage.
DESCRIPTION OF THE FIGURES
FIG. 1A is a schematic diagram (side view) illustrating an embodiment of a device for CDEB based manufacturing of defined micro structures such as holes, consisting of the insulating substrate material to be structured (1) between electrodes (2); the electrodes can have various forms (2) and distances to the substrate material; the electrodes are connected to an adjustable and process controlled high voltage source (3); the latter consists of an adjustable voltage source (3A) receiving feedback (3C) from a current monitor (3B) that modifies, that is, usually disables, voltage source output after a preset delay (delay usually zero or near zero) once a specified trans-substrate current (or current pattern) has been reached (DEB onset). Trigger level and voltage source properties are usually set or programmed by the operator. An optional series resistance R (4) may be connected in series with the electrodes to limit the current during CDEB. A series resistor is particularly useful when only very small currents are permissible for substrate perforation and stray capacitances and/or timing of the voltage source render the precise current control difficult and imprecise, respectively. In the most basic CDEB set-up, the voltage source usually controls the CDEB process in such a way that the maximum current and the duration of current flow after DEB onset is adjusted. DEB onset can be detected in various ways; most suitably, DEB is usually detected by a trigger monitoring the trans-substrate current. In a preferred embodiment, a steep increase in this current by usually more than one order of magnitude indicates DEB onset. The substrate material and electrodes may be surrounded by a controlled gas composition and pressure (5).
FIG. 1B illustrates an embodiment of a device for extrinsically heat supported CDEB. Heat is supplied by a heating filament (6) controlling the substrate temperature. For simplicity, the heating filament also serves as counter electrode (6). In this example, the electrode (6) is directly heated by an electric current applied to terminals (7). For practical reasons the heated electrode was connected to ground while the opposite electrode supplied the voltage (“hot” end). The electrode (6) can also be indirectly heated by surrounding the electrode with a suitable heating element. One realisation used to produce holes of 1-10 um diameter in Menzel S1 cover slides consisted of a feedback controlled 0-30 kV, 0-300 uA voltage source, connected to a 0.1-2 mm Pt-wire electrode on one side of the slide (distance ca. 0.1-0.8 mm) and connected to a Pt heating filament (1×1 mm2 active area) ca. 0.05-0.5 mm from the other side of the slide. During perforation the cover slide was mounted on a perforated glass/ceramic slide allowing electrode access to the cover slide from both sides. The voltage source (3) was controlled according to the description of FIG. 1A. The CDEB voltage amplitude was chosen not to cause DEB on its own; DEB was initiated by the application of a short but controlled heating voltage/current to terminals (7), the latter usually automatically applied after reaching a preset voltage as part of a microprocessor based process control. A typical I-V time course of a perforation is shown in FIG. 3E.
FIG. 1C illustrates an embodiment of a device for extrinsically heat supported DEB. The substrate temperature is locally controlled by a laser (8, beam indicated as dashed line). Additionally, a pyrometer can be used to supply feedback to the laser for precise substrate temperature adjustment. After CDEB voltage application, DEB is initiated by a short 1 laser pulse. The voltage source (3) is controlled according to the description of FIG. 1A.
FIG. 1D illustrates an embodiment of a device for extrinsically heat supported DEB based on a device as in FIG. 1A (resistance omitted for simplicity) with a modified electrode (2). One electrode (2) is replaced by a burner (9) focussing a flame (10) onto the substrate surface. Undesired global heating and deformation of the substrate can be avoided by heat shields (11), e.g. Schott CERAN™ plates, providing only restricted access to the substrate surface (usually recommended is a second heat shield (11) on top of (1) avoiding heat caused deformations of (1)). If the flame outlet of the burner is metallic it can be directly connected to the feedback controlled high voltage DEB source (3). Otherwise the original electrode (2, lower electrode in FIG. 1A) must be placed in the flame or near the DEB location. Asymmetric heating of the substrate surface (i.e. one sided heating) leads to asymmetric holes (FIG. 3B). After CDEB voltage application, DEB is initiated by a short contact between flame and the substrate region to perforate. The voltage source (3) is controlled according to the description of FIG. 1A.
FIG. 2A is a schematic diagram illustrating a possible embodiment of a current-voltage source for formation of CDEB structures, such as holes for carriers of biological membranes. The operator (1) sets via a computer (2) with attached digital-analog/analog-digital converter (3) the voltage (4) and maximum current (4) of the controllable high voltage source (6) (e.g. EuroTest CPP300304245, Germany). Voltage is applied to the carrier (9) via electrodes (8) and an optional current limiting resistor (7). The resistor may be necessary when the internal current limitation of the voltage source is not precise or does not respond quickly enough for some substrates or large capacitances in parallel to the electrodes render the current limitation circuits of the voltage source inefficient for quick response. The current through the substrate (9) is monitored by the computer via a current monitoring signal (5) coming from a current monitor, which may be part of the voltage source. Upon beginning of the dielectric breakdown a timer is triggered that sets the duration of the controlled current flow. This consequently sets the electric energy at a given voltage, which is partially transformed into heat energy, driving the actual hole forming process. For many substrate materials, e.g. glass, the current flow interval after DEB detection can be set to zero.
FIG. 2B is a schematic diagram illustrating a possible embodiment of a current-voltage source for intrinsically heat supported CDEB based on AC voltages only. The DEB voltage electrodes (2) are connected to ground and the output of a high voltage transformer (3, e.g. flyback transformer without rectifier from CRT type monitor), respectively. The transformer output is also grounded via a resistor (4), serving as trans-substrate current monitor. The transformer is driven via a transistor (5, e.g. IGBT or power npn). The transistor is driven by pulse trains usually received from a computer controlled AD/DA converter. Upon DEB onset, the increased trans-substrate current leads to an increased voltage drop across (4) which is sensed by the trigger (8). The trigger signal (activ=Low!) disables via an AND gate (7) the transistor (5) and consequently further high voltage generation, even if the computer did not yet process the trigger signal (10). The voltage drop across (4) and consequently trans-substrate current at which high voltage generation is stopped is set by the trigger level line (12). The trigger compares the voltages across (4) and (12) (e.g. using the LM393 comparator) and consequently sets an internal RS flip-flop which disables (5) via (7) until reset (i.e. trigger output=High) by the operator/computer via (11).
FIG. 3A shows a microscopic image (upper picture) of a hole produced with CDEB in a 20 um thick polypropylene (PP) sheet. The hole diameter is ca. 5 um (aspect ratio ca. 4). The lower part shows the current-voltage curve (uA-kV) recorded while the trans-substrate voltage was increased until DEB occurred. The parameters were: R=10 GOhm, V_DEB=6.4 kV, I_trigger=1.8 uA and the voltage was raised with dV/dt=60V/80 ms. Voltage was lowered to 0 kV immediately upon detection of DEB at ca. 6.4 kV. Electrode distances to the PP sheet were ca. 10-200 um.
FIG. 3B shows microscopic images of holes produced with thermally supported CDEB (according to FIG. 1D) in a ca. 170 um thick glass cover slide. The hole diameter is ca. 3 um. The parameters were: V=20 kV, I_trigger=40 uA, flame source butane micro torch with flame touching substrate until DEB. Voltage and flame were turned off immediately after DEB onset. Electrode distance to the glass substrate surface was 300 um. The micro torch metallic flame outlet was connected to the voltage source. Upper picture: torch side of the substrate/hole; lower picture: opposite side of the substrate/hole.
FIG. 3C shows electron microscopic images of the heating filament side of holes produced with thermally supported CDEB (according to FIG. 1B) in a ca. 170 um thick glass cover slide (Menzel S1) at different magnifications (upper panel 1500×/lower panel 5000×, scale bar see figure). At 1500×, glass filaments ejected during CDEB and now covering the substrate surface are visible.
FIG. 3D shows an electron microscopic image of the voltage electrode side of holes produced with thermally supported DEB (according to FIG. 1B) in a ca. 170 um thick glass cover slide (Menzel S1, scale bar see figure).
FIG. 3E shows the time course of the current (3)-voltage (2) relationship during the thermally supported perforation of a standard microscopic cover slide (Menzel S1 20×20) with a pure DC voltage (vertical axis indicating kV and 30*uA, i.e. maximum substrate current shown is 600 uA, horizontal axis in milliseconds). After voltage application, the DEB process is initiated by a short heating pulse using a Pt filament (ca. 1×1 mm2 active area parallel to the slide surface) mounted c lose (ca. 0.3 mm) to the slide surface. The heating filament serves also as ground electrode. The voltage electrode was mounted ca 0.5 mm from the cover slide opposite the ground electrode. The current heating the filament is indicated (1). The filament heating current interval was preset; the DC voltage of ca. 12 kV was shut-down immediately after dielectric breakdown detection. The trigger signal used for this shut-down was a sudden raise of several orders of magnitude of the substrate current (3) accompanying the hole formation (see figure at ca. 530 ms). The hole was ca. 3 um in diameter (aspect ratio ca. 50). This cover slide was used in a patch clamp setup and produced a giga seal with Jurkat-cells in Ringer solution within less than 5 sec after moving of a cell over the hole by suction. The optimal heating current was determined experimentally and is shown in arbitrary units; heat produced by the Pt-filament was controlled by the duty cycle of the 5V/10 kHz heating power supply.
FIG. 3F depicts the conditions during a combined AC-DC voltage produced CDEB. After application of the DC voltage (2), an additional AC voltage source in series with the DC supply is activated, providing an AC voltage of ca 10,000-30,000 V, f=15 kHz, duty cycle=0.4 (the current in Ampere supplied to the primary coil of the AC supply high voltage transformer is shown (1)). The AC voltage was supplied intermittently (two series of 10 pulses of 60 and 40 ms (2nd series shown), respectively, with 5 ms between AC pulses; pulse 10 not applied because of trigger signal induced AC and DC shut down) to better control the substrate heating process, thus avoiding micro cracks in the substrate caused by mechanical tensions. The AC current induced heating of the substrate leads to a dielectric breakdown during which substrate material is ejected leading to a ca. 3 um hole (aspect ratio ca. 50) and usually the deposition of thin filaments ejected from the hole on the substrate surface. A strong increase in AC current during DEB was used as trigger signal for immediate AC and DC voltage shut down to avoid closing the hole by excess molten glass. DEB also leads to a typical increase in DC current (3). Substrate: Cover slide (Menzel S1 18×18). The horizontal axis is in milliseconds, the vertical axis shows V in kV units and current in uA*30, the maximum substrate current (DC component) shown is 600 uA.
FIG. 3G depicts the time course of a hole formation by intrinsically heat supported CDEB (AC only) in a standard microscopic cover slide (Menzel S1). The AC current flowing through the primary coil of a high voltage transformer during substrate structuring is shown (1). The current flow as well as electric arcs between the electrodes and the substrate as well as dielectric losses inside the substrate lead to a fast (nearly approaching exponential growth) temperature increase causing melting, evaporation and ejection of substrate material out of the forming hole. This material can be found as thin filaments at the substrate surface. To avoid closing the hole again with excessively molten glass, the AC voltage is immediately turned off after hole formation. Parameter: f=20 kHz, duty cycle 0.65, AC voltage amplitude ca. 10,000-30,000 V, 40 AC voltage pulses a 10 ms with 10 ms off-time in between preset; pulse application was stopped immediately upon hole formation, which was indicated by a strong increase in trans-substrate current as well as increase of the current through the primary transformer coil; pointed Pd-electrodes were used with a distance of ca. 0.5 to 1 mm to substrate surface. Hole sizes under these conditions ranged between 1-5 um (aspect ratio between 120-30). The horizontal axis is in milliseconds, the vertical axis in Ampere.
FIG. 4 shows a possible realisation of a device using CDEB micro structured carriers for electrical membrane measurements. The carrier (1) separates two fluid compartments having any shape and boundaries (8, 9) which are only connected through the carrier channel (2) formed by CDEB. One side of the channel is covered by a biological membrane (3). Upon tight binding of the biological membrane such as an artificial unilamellar lipid bilayer to the carrier surface, voltages applied through the fluid immersed (redox) electrodes (4) lead to a current that is only dependent on the properties of the biological membrane itself. Current voltage measurements may be performed with a suitable device (5) allowing to set the voltage (6) and measure the current (7). For some electrophysiological measurements the device (5) may be substituted with a voltage measuring device.
FIG. 5 shows a possible realisation of a device using CDEB micro structured carriers for electrical membrane measurements on biological cells such as patch clamp measurements. The carrier (1) separates two fluid compartments (6, 7) which are only connected through the CDEB produced channel (2). One side of the channel is covered by a biological cell (3). Upon tight binding of the biological cell to the carrier surface, voltages applied through the fluid immersed (redox) electrodes (4) lead to a current that is only dependent on the properties of the cell membrane. Upon removal of the membrane patch covering the hole, the almost entire remaining cell membrane contributes to the trans-carrier current (whole cell mode). Current voltage measurements may be performed with a suitable device (5), such as a patch clamp amplifier (e.g. Axon Instruments).
FIG. 6 illustrates the sealing process of a K562 cell to a CDEB produced carrier used in a patch clamp configuration (upper panel) and subsequent single channel recordings in cell attached mode (lower panel). Menzel S1 cover slides were perforated using an intrinsically heat supported CDEB process (AC only, train of 4 pulses with 200 ms duration separated by 100 ms off-time, V ca. 20,000 V at 40 kHz, trigger current (here, the current through the primary coil of the high voltage transformer was monitored) was linearly raised form 2000 mA (first pulse) to 2400 mA (last pulse), pointed palladium electrodes with 2.5 mm electrode distance; the slide was tempered after hole formation by short (ca. 0.5-3 sec) heating with a micro torch flame) and inserted into a set up similar to FIG. 5 (electrodes Ag/AgCl) but allowing for an air pressure reduction at the lower compartment. Buffer volumes were ca. 10̂−5 L, buffer used was Krebs. Carrier access resistances ranged from 6 to 12 MOhm. For cell measurements, ca. 3*10−6 L of a cell suspension (10̂7/ml cell density) were added to the upper compartment; cells were positioned by reduction of the ambient air pressure at the lower compartment by ca. 1-50 mbar. Cell positioning usually occurred within 0-20 sec after cell addition; in the upper panel, the sealing process is monitored by application of a voltage V=+60 mV using the Ag/AgCl electrodes. The decrease in leakage current indicates the progressing sealing process. Final seal resistance was ca. 4 Gigaohm. Subsequent application of a voltage of V=−30 mV revealed the activity of membrane channels in cell attached mode (lower panel). All voltage clamp and current recordings were performed using an amplifier Axopatch 1D (Axon Instruments) with a filter frequency of 5 kHz using the build in filter and the Fetchex (Axon Instruments) software.
Patent Application
20080047935
