Patent Publication Number: US-2006006061-A1

Title: Electrical microhydraulic multiplex system and use thereof

Description:
TECHNICAL FIELD OF APPLICATION  
      The invention describes a device and several methods for the highly parallel, electrically controllable processing of molecules (especially biopolymers) in an integrated microstructured hybrid component (combination of microfluidics and electronics). The controlling is mediated via electrodes by the known electrophoretic drift of molecules in an electric field. This way of controlling, however, is designed to the effect that the active control of the molecular movement in different units (e.g. channels) of the microfluidics can be carried out in a highly parallel manner independent for each unit.  
      This offers the possibility of new approaches in tasks which require a high sample throughput and/or a larger number of selectively used processing steps. Applications are possible in the fields of biochemistry and molecular biology, molecular diagnostics, synthesis and analysis of novel agents, and biomolecular information processing. Typical applications are found in the pharmaceutical industry, the automation of laboratories, clinical diagnostics, process and environmental technology, evolutionary biotechnology and adaptive nanotechnology.  
     THE STATE OF THE ART, DISADVANTAGES OF THE STATE OF THE ART  
      The electric transport control of biomolecules by use of a moderate number of electrodes (&lt;1000) belongs to the current state of the art. Particularly in electrophoresis (e.g. gel electrophoresis and capillary electrophoresis), considerably less electrodes are driven individually and analogously, inclusive of voltage and current monitoring. In microstructures, electrolysis will limit the maximum allowable potential value at the electrodes due to gas generation. The use of gels for better discrimination during transport entails problems because of restricted reusability and cross contamination.  
      The effect of electrodes on the transport of biomolecules is heavily affected due to shielding caused free charge carriers in the solution. If an electric field is applied between the electrodes in a solution, in the presence of charged particles an electric double layer forms which, depending on the type and concentration of the particles, will be extended and fixed to a higher or lesser degree. A difference is seen between a rigid and a diffuse double layer. In case of a high concentration particularly of strong electrolytes, the electrodes will be massively shielded, resulting in a considerable reduction of the field in the interior of the solution so that the effect of the field on the particles (e.g. for the purpose of transporting these) is heavily decreased. Strictly speaking, however, these processes pertain only to the case of equilibrium, i.e. when all particles have been oriented and there is no noteworthy passage (exchange current density) through the double layers. Although research has been directed to such kinetic effects, their beneficial use as part of a dynamic electric control system has still been neglected.  
      Some present-day microreactors already use electric fields to convey molecules to specific sites (DNA chips), or to prevent them from diffusing away from a site. Already prior to use, these sites are provided with specific substances, e.g. short DNA strings, to thus allow for conclusions on the substances contained in the liquid on the basis of the site information. Active movements of molecules in solutions have been described (e.g. by Fuhr) but are not yet economically utilizable. In the field of electrophoresis in microstructures, numerous efforts are under way to divide molecule mixtures by use of impeded movement in gels and to thus make specific molecules available for further processing: here, however, the reactors, arranged in parallel, are not controlled individually. Further documented uses of electric fields relate to electroporation and the sorting of cells. In all of these examples, there is reached only a limited parallelism of the control.  
      M. Heller describes electrically configurable DNA chips with electrically improved molecular discrimination. Apart form their small number of electrodes, a disadvantage of these DNA chips resides in the limited lifespan of the coated electrodes. The molecule processes which up to now have been performed in the flow with active movement suffer from the disadvantage of the merely small number of electrodes, which is typically below 100. Such small numbers result from the fact that these electrodes have exactly set analog voltage potentials assigned to them in order to a) allow for an optimum traction of the molecules and b) to prevent the generation of gases by electrolysis. The potentiostats used for this purpose are expensive and bulky.  
      More recent efforts aimed at the use of integrated electronics for the sensorics and control of biochips (e.g. Infineon) are expensive due to the required complex circuits and thus are not yet as highly parallel as necessitated in biotechnology.  
     Object  
      It is an object of the invention to make it possible to use a limited number of control lines in order to control a considerably larger number of electrodes for the purpose of highly parallel control of molecules in microfluidics reaction systems.  
      Solution  
      According to the invention, the above object is achieved by a microfluidics system as set out in claim  1 . Modifications of the invention are mentioned in the subclaims.  
      Thus, according to the invention, there is proposed a special type of arrangement of electrically interconnected electrodes, wherein each group of the thus interconnected electrodes has an output of the control electronics assigned thereto. When a control signal is applied to this output, this control signal will be present on all electrodes of this group. The arrangement of electrodes of two respective groups is selected in such a manner that both groups together include at least one pair of electrodes, with the distance of the electrodes being minimum. When control signals are applied to both groups of electrodes, an electric field will be generated between the electrodes of the above mentioned pair of electrodes, which field has a sufficient strength to cause the desired manipulation (e.g. the transport of a molecule). For all other electrode pairs of these two groups of electrodes, it holds good that, if the distance of the electrodes of the respective pairs is larger than the minimum distance, the electric field has merely a strength which is unsuited for performing a manipulation.  
      By suitable selection of the composition of the liquid (particularly the ions in the liquid are decisive), it can be accomplished that the electric field in the liquid will be reduced only with a delay. This means that the electric field will still continue to exist for a certain time after the control signals have been switched off. The interval at which the pulse-shaped control signals follow each other at different outputs of the control electronics can now be selected to be shorter than the relaxation time of the electric field. In this manner, there is carried out a time-multiplexed control of individual electrode pairs and thus a quasi-parallel manipulation of a plurality of molecules within the microfluidics system so that, on the whole, (with corresponding scaling) larger quantities of molecules of one or a multitude of types can be controlled in a massively parallel manner.  
      Basic Features of the Solution  
      a) Digital voltage  
      Normally, the output of a digital component can assume only three states: 0, 1, tristate →Z, i.e. about 0 Volts, 3.3 or 5 Volts (V cc ) or high-ohmic (inactive). However, the control for a pair of electrodes in a solution with charged molecules represents—under the aspect of electricity—an arrangement comprising at least resistors (the conduction is performed via charge carriers existing in the liquid, normally ions) and capacitors (local barrier layers may build up, which will then further increase the already existing capacities). As a result of this fact, by temporally correct switch-on and switch-off of the digital outputs at the electrodes, virtually every desired voltage can be set for controlling the molecules (low-pass behavior, D/A converter). By utilizing this effect, the technical expenditure for control is considerably reduced. Thus, the actually effective electric field between two electrodes controlled in this way can vary in strength and direction almost in any desired manner between zero and the maximum. Correspondingly, also the effect of the field on the molecules can be set in virtually every desired manner.  
      b) Due to the inherent charge of the molecules or the adhering charge carriers, the forces which can be exerted on these molecules will increase along with the field strengths perceived by the molecules. However, the maximum field strengths are limited because of the separation of water molecules into their components. Pulsating fields, however (the actual field is applied to the electrodes only for a short time), nonetheless make it possible to drive the molecules with higher field strengths. The duty cycle is settable. Thereby, if a corresponding setting is carried out, a generation of gas bubbles at the electrodes is avoided. The gas still produced as a result of the electrolysis is given time to allow diffusion thereof from the electrodes into the solution and will not cause disturbances. For the establishing of the electrode geometries, the inhomogeneities occurring at the edges of the electrodes will in this context have the positive effect that the voltages are very small and thus can remain within the voltage ranges of V cc  given by present-day digital technology.  
      c) 2-dimensional electrode time multiplexing  
      By the use of highly integrated commercial digital components comprising several hundreds of output drivers (n output drivers), it now becomes possible to drive electrode bundles in the magnitude of n 2  independently from each other and thereby provide several thousands of reaction chambers with molecules or to remove molecules from the reaction chambers in any desired manner. In this regard, it is irrelevant whether these reaction chambers are formed as spatial chambers or merely are generated spatially-temporally within moving liquid streams. Because of the ionic double layers which are in the process of being generated and because of the associated field shielding, the electrode control processes can be interleaved in time in such a manner (similar to the annular-core-type storage devices from the early years of computer technology) that several thousands of electrodes can be operated although merely several hundreds of active drivers of digital components exist. Using this method, only 200 actively driven conduits are needed for a 100 ×100 field of electrodes. Only in situations where electrodes of two serial electrode circuits connected to different driver outputs are arranged in close proximity to each other (in respectively two serial electrode circuits, this situation exists at least for one electrode pair), the fields (e.g. due to inhomogeneities at the edges of the electrodes) are strong enough to move the molecules locally existing in this area. The relaxation time of the double layer can be in the range of milliseconds, depending on the puffer solution. Only thereafter, the electrode potential will have been eliminated or built up. During such times, consequently, other electrodes can be driven for particle transport or manipulation (multiplexing). In the process, the particle types of the buffer, which are contained in solutions, will be compiled according to the given task.  
      d) Direct integration of logic elements into the electrodes  
      Depending on the existence of the technology or a sufficiently low cost level, the output drivers of the digital components can be integrated directly into the carrier material. This will be advisable especially if silicon is used as a carrier material. The silicon surface required for the additional logic inclusive of the corresponding drivers can be easily made available among the electrodes. By means of sophisticated bus topographies and mixing hierarchies—for instance, a small surface is driven by the above described multiplex method and a large number of such small surfaces is distributed on the whole module—the degree of integration can be even further improved. However, it is to be considered that these microstructured bioreactors are disposable articles and thus should be inexpensive in manufacture.  
      Improvements and advantages over the state of the art  
      The advantages achievable with the invention relate to the now possible highly parallel integration of thousands of electrodes and the thus accomplished availability of a combinatorial variety in the reaction paths. First, the described invention makes it possible to carry out, within a microfluidics system, a large number (10 3 -10 6 ) of different reactions or separation methods with parallel and mutually independent control. Apart from the use for this direct, massively parallel reaction control, the highly parallel electrode control can be utilized to configure—i.e. to program—a microfluidics system for various purposes. Further, the desired molecular processing need not be known in advance and need not be identical for all samples but can be made dependent from intermediate results. The now possible provision of thousands of independently controlled electrodes will open up novel fields of application in chemical or biochemical reaction systems, e.g. in analytics and in agent synthesis. This wide variety of reaction paths cannot be realized in microstructures under classical circumstances because an individual flow control would be prohibited by the comparatively large size of the valves required. By this massive parallelism, biochemistry is rendered programmable, and all tools of informatics (e.g. data flow architectures) become useful also for direct chemical conversion.  
      1. Work is performed by use of individually pulsed “digital fields”.  
      2. The pulse and duty cycles can be adapted to the particle types so that “electric filters” and “amplifiers” can be produced which allow for a preferred transportation of specific particle types.  
      3. Depending on the electrode design and the field strength, the fields can be formed to be very inhomogeneous so that also molecules which have dipole characteristics can be transported and manipulated.  
      4. The duration, the frequencies and the duty cycles of the applied digital potentials allow not only for the avoidance of electrolysis effects on the electrodes but also allow for the above mentioned multiplex operation, notably because of the relaxation behavior of mobile charge carriers in the solution. 
    
    
     EXAMPLES OF AN EMBODIMENT  
      In  FIG. 1 , left-hand part, there is shown a matrix with respectively six traces in the x- and y-directions (x 1 -x 6  and y 1 -y 6 ). Liquid channels can be laid diagonally across the 36 electrodes driven by these traces. The electrodes have rectangular pulses assigned thereto. A plurality of reference electrodes distributed in the liquid channel have assigned thereto a rectangular voltage of a higher frequency which is selected to the effect that the medium voltage level will be set e.g. exactly at V cc /2. By setting the operating point in this manner, it becomes possible to generate various difference voltages by use of only two voltages (0 V and V cc ). By a well-aimed order of the impulses, charged molecules can be controllably moved through attraction and repulsion. Shown on the right-hand side is a possible pulse sequence by which the electrodes can be driven individually. As outlined already above, the electric behavior of the liquids in the microstructure plays an important role in the utilization of the relaxation properties of the ionic double layers (ca. 10-20 ms) Without these double layers, the typical relaxation times are in the nanosecond range and thus are too short for the utilization of this effect.  
       FIG. 2  shows a possible realization of the above two-dimensional array. The electrodes are connected diagonally in order to allow for horizontal and vertical channel structures. The electrode connections are configured to the effect that electrodes which are no direct neighbors are arranged at least four electrodes apart from each other. The fields which then act across the four electrodes can be neglected because of the strong decrease of the field strengths. Additionally, by well-aimed control, it can be accomplished in many cases that even the more remote electrode pairs, although active, will have no influence at all due to the absence of molecules. Nonetheless, of course, it should be kept in mind that the reasonable number of simultaneously active electrode pairs must be much smaller than x y since the forces integrally exerted on the biomolecules would otherwise become too small. 
    
    
      The design illustrated in  FIG. 2  is however subject to the precondition that a two-layered trace layout is allowable. It is of no relevance whether these two layers are arranged on the selfsame side of the basic material (e.g. silicone) or each of the sides is provided with a layer comprising the corresponding vias.  FIG. 3  shows a test example for the case that only a one-layered trace layout is possible. However, the scaling work suffers considerably from this restriction to a merely one-layered trace layout and can be reasonably performed only in one dimension.