Abstract:
Microtechnologically prepared component as a flow cytometer. The component contains a preparation area to specifically influence and separate the particles, preferably by dielectrophoresis, a measuring channel area for characterizing the particles, and a sorting area for sorting the particles identified in the measuring channel area by dielectrophoresis. The sorting includes switching elements which permit active guidance of the particles into two or more subchannels corresponding to the criteria which have been registered in the measuring channel area. With a component configured in this way for the use of a flow cytometer, quick and precise sorting of particles, in particular biological cells in a suspension, can be implemented.

Description:
BACKGROUND OF THE INVENTION 
     The present invention relates to a microfluidic component comprising a substrate having a channel for leading individual particles through for sorting particles in a fluid flow, in particular a liquid flow, and also a method for sorting particles. 
     The manipulation of suspended particles is generally known and is described, for example, by G. Fuhr et al in “Naturwissenschaften” [Natural sciences] volume 81, 1994, page 528 ff. Here, the suspended particles can be any desired particles or else biological cells. In the following text, therefore, in general only particles will be mentioned. The microsystems form in particular channel structures through which a suspension liquid with the particles to be manipulated flow. In the channel structures, microelectrodes are fitted to the channel walls and have high-frequency electric fields applied to them. Under the action of the high-frequency electric fields between two electrodes, polarization forces are produced in the suspended particles on the basis of negative or positive dielectrophoresis, said forces permitting repulsion or attraction by the electrodes and, in interaction with flow forces in the suspension liquid, permitting manipulation of the particles in the channel. Electrodes of this type are described, for example, in WO 00 00 293. 
     In S. Gawad et al, “1st Annual International IEEE-EMBS Special Topic Conference on Microtechnologies in Medicine &amp; Biology Oct. 12-14, 2000, Lyons, FRANCE:”, pages 1 to 5, and also from S. Gawad et al “Lab on a Chip”, 2001, 1, pages 76 to 82, it is known to analyze cells in a microchannel by means of impedance spectroscopy. In this case, use is made of electrodes embedded in the channel, so that at least two field areas are produced, in which an impedance measurement is carried out. Evaluation is then carried out by means of a difference measurement, for example by means of a measuring bridge. With regard to further details, reference is made to these publications and the explanations given therein relating to the measuring principle and to the prior art. 
     The results of such an analysis can then be used to carry out cell sorting next. Here, it is likewise known to carry out cell sorting on the basis of dielectrophoresis. For example, it is known from U.S. Pat. No. 4,326,934 to extract sulfur from oil by means of dielectrophoresis. 
     U.S. Pat. No. 5,489,506 discloses a method and an apparatus for the continuous sorting of living cells from a mixture, in which the cells passing one or more electrodes are sorted out in accordance with their size, physical construction, chemical composition and electronic characteristics, the electrodes being connected to radio-frequency generators. 
     In the method described above, the different electrophoretic characteristics for the particles are used to lead these out of the fluid flow. This is passive sorting, since a specific decision as to whether the particles are to be led out or not cannot be made. 
     The previously known devices for characterizing particles, in particular biological cells, sorting and counting (cytometers) are distinguished by very complicated devices, which generally comprise an optical analysis device based on FACS (fluorescence-activated cell sorting) or a combination of optical and electrical measuring devices. Purely electrical detection and sorting is not carried out. Therefore, in the case of these devices, additional preparation of the particles for the optical analysis is required. This makes the analysis by means of such devices complicated and time-consuming. 
     It is an object of the present invention to provide an electrical flow cytometer which, on a substrate in micro- or nanotechnology, permits the identification and counting and sorting as quickly and accurately as possible. 
     SUMMARY OF THE INVENTION 
     According to the invention, the foregoing object is achieved by a microfluidic component comprising a substrate (a) at least one channel ( 2 ) for leading through individual particles ( 17 ) in a fluid, the channel ( 2 ) comprising: a preparation area ( 6 ) to specifically influence and separate the particles ( 17 ), a measuring channel area ( 7 ) having electrode devices for characterizing the particles ( 17 ), and a sorting area ( 8 ) having electrode devices for sorting the particles ( 17 ) identified in the measuring channel area ( 7 ) by means of dielectrophoresis; and (b) conductor tracks ( 12 ) which are electrically connected to the electrodes ( 13 ,  14 ′,  14 ″,  20 - 30 ) in the individual areas ( 6 - 8 ) in order to transmit signals to the electrodes and signals from the electrodes and a method which comprises. 
     The basic idea of the invention is that, on a carrier material, called a substrate below, in at least one elongate channel through which the individual particles are led in a fluid, said particles are prepared firstly in a first area in the fluid flow for the subsequent measurement in a second measuring channel area and then, after leaving the measuring channel area, the particles are separated in a third area, the sorting area, on the basis of the registered characteristics of the particles. In this case, it is of course possible for the separation to be performed into more than just two different types. Accordingly, the channel comprises a preparation area with first electrode devices to specifically influence and separate the particles, preferably by means of dielectrophoresis. This is followed by the measuring channel area having second electrode devices for characterizing the particles. There then follows the sorting area having third electrode devices for sorting the particles identified in the measuring channel area by means of dielectrophoresis. Of course, on the substrate there are additionally conductor tracks which are electrically connected to the respective electrodes in order to transmit signals to the electrodes and away from the electrodes. For this purpose, the component must be connected to appropriate measurement and control devices, which are not the subject of the invention. 
     In this case, the cross sections of the individual areas vary in such a way that the cross section of the channel (measuring channel) in the measuring channel area is substantially smaller than the cross section in the preparation area and in the sorting area. 
     The electrode arrangements used in connection with dielectrophoresis always comprise a pair of identical electrodes which, as a rule, are fitted to the channel walls laterally or at top and bottom, so that in the coincident areas between the individual electrodes an electric field can build up which causes the particles to make the desired movements. In the following text, therefore, the terms electrodes or electrode pair will be used as synonyms. 
     In order that characterization and subsequent sorting is possible at all, it is necessary in the preparation area to separate the particles, which may possibly cohere in clusters, and then to feed them in a controlled manner to the measuring channel area. This is preferably done by means of corresponding electrode arrangements, so that the particles are separated by means of dielectrophoresis and the forces acting on the particles as a result, and are brought into the appropriate particle path. 
     According to a preferred development, the electrode devices for separating the particles have electrode arrangements which contain first electrodes arranged obliquely with respect to the flow direction and having a substantially funnel-like arrangement and, following these in the flow direction, spaced-apart parallel second electrodes in a likewise funnel-like arrangement with a passage opening. In this case, the following parallel second electrodes are kept continuously at potential, so that incoming particles are held up and can pass through the passage opening only if they pass through individually. Larger clusters cannot pass through. As a result of the configuration, the passage opening constitutes a narrow slit in the channel. In order to separate the clusters located upstream of the passage opening, the first electrodes are pulsed, so that, on account of the forces which occur as a result, the individual particles attempt to escape and in the process are detached from the other particles. The distance between the electrodes is preferably two to four times the particle diameter. 
     Following this, the particles are aligned in the fluid flow, so that the particles move on a defined particle path. According to a further preferred embodiment, the first electrode arrangement for separating is directly followed by a second electrode arrangement for aligning the particles onto a defined particle path. The separation electrode arrangement is preferably also combined with the electrode arrangement for alignment, in which the previously mentioned second parallel electrodes having the passage opening have lengthened parallel electrode arms at the spacing of the width of the passage opening (gap). This electrode arrangement therefore comprises two upper electrode arms arranged at the spacing of the gap width and two associated lower electrode arms, in each case a field building up on an associated upper and lower electrode. As a result of arranging four electrode arms, the particles attempt to find the equilibrium state exactly at the center. In this case, it is necessary for the length of the electrode arms, in relation to the flow velocity of the solution, to be sufficiently long in order that the equilibrium state can be reached. The width of the passage opening and the spacing of the electrode arms is slightly greater than the particles. 
     These particles prepared in this way then pass into the measuring channel area, which preferably has a cross section which is only slightly greater than the particles flowing through. Its typical channel size can be 5 to 10 μm. The passage of a particle, in particular for example of an individual cell, is registered and identified by the change in the electrical impedance which, in the case of a cell, has a relationship with the characteristics of the cell size, cell membrane and the cytoplasma. The above cited article by Gawad et al., is incorporated by reference. 
     According to another preferred embodiment, a wide measuring channel is used, which has the advantage that the risk of a blockage is reduced. In this wider measuring channel, the individual particles are deflected onto a specific path on one side of the channel by means of dielectrophoresis and then pass through the measuring field between two electrodes, the reference field being arranged in the area in which no particles move. 
     The dimensions of the channel lie in the order of magnitude of the typical particle size to be measured or an order of magnitude above. The electrodes can be arranged in various ways on the walls, in order to provide at least two detection areas in the same channel. The electrodes can be arranged one after another on one side of the channel, circling around the channel, only above, or above and below or laterally. If the electrodes are arranged one after another only on one side of the channel, there is a great influence on the particle position during the measurement. If the position of the particles is to be determined accurately, it is expedient to perform the measurement by means of a differential measurement between an offset upper and lower electrode pair. This has the effect that, depending on the vertical alignment of the particle, the upper or lower electrodes are influenced more or less. In a corresponding way, this can be carried out on the side walls. 
     After the individual particles have passed through the measuring channel area, they come into the sorting electrode area having a larger cross section. In order that proper sorting is possible, according to a preferred embodiment, there is upstream of the sorting electrode arrangement an electrode arrangement for aligning the particles, in order to lead the latter onto the narrowest possible path in the fluid flow. This permits exact sorting at high speed, since the corresponding electrodes can have a small electrode length and therefore the path of the sorted particles led out of the particle path is short along the electrodes, and therefore a new selection can be made relatively quickly thereafter. 
     According to a particularly preferred refinement of the invention, the electrode devices in the sorting area have a first sorting electrode arrangement and at least a second sorting electrode arrangement, the first sorting electrode arrangement being arranged in the path of the particles and the second sorting electrode arrangement being arranged at the side of the paths of the particles in order specifically to lead particles sorted out by the first sorting electrode arrangement onward. The second sorting electrode arrangement preferably has an output to pass the particle, so that the particles sorted out move on a second path, substantially parallel to the first particle path. Following this, a fork is arranged in the channel, which divides the fluid flow in accordance with the particle paths. The substrate therefore has one inlet and at least two outlets. The outlets can then be fed to appropriate holding containers or the like. 
     In order that observation and detection by means of optical methods is also possible, it is expedient to design the substrate to be transparent. 
     The substrate according to the invention therefore permits, in the smallest possible space, the determination of individual particles from a large number of particles passing through, of the order of magnitude of 100 or more particles per second. The invention permits the positioning of the particles, the measurement of the impedance, the velocity or the position of the particles flowing in the measuring channel, and a flow switching arrangement in the sorting area, which allows the particles to be led into two or more subchannels in accordance with the previously determined criteria in the measuring area. Such a microtechnological produced component can be used to count cells, to distinguish them and to sort them in accordance with their type, their size, their cell membrane characteristics, the presence and/or the activity of specific membrane receptors. Within the context of the sorting, the identification and counting of particles is therefore also possible. 
     According to the method for sorting particles in a fluid flow by means of a microfluidic component, the following steps are carried out: 
     A separating the particles by means of dielectrophoresis and subsequently positioning the particles in the physical center of the fluid flow, 
     B characterizing the separated particles into a narrow fluid flow in a measuring channel area by means of impedance measurement, 
     C sorting the particles registered in the measuring channel area on the basis of the characteristics determined in B by means of dielectrophoresis by actively sorting particles out of the particle path and specifically leading the particles sorted out onward in a second particle path, which runs substantially parallel to the first particle path, 
     D dividing the fluid flow corresponding to the particle paths and 
     E if necessary, repeating steps C and D. 
     If further subdivision and sorting is to be carried out, steps C and D are repeated appropriately often. 
     In order to generate the electric fields, preferably one or different voltages with a frequency from 1 kHz to 200 MHz, expediently with peak-to-peak voltages of at most 2 V, are used. 
     Before sorting, the velocity of the particles is advantageously measured optically or electrically. 
     In principle, it is possible to carry out the method by means of devices arranged and integrated on the component or else with external devices for field production. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       In order to illustrate the invention further, the latter will be explained in more detail below by using exemplary embodiments in conjunction with the accompanying figures, in which: 
         FIG. 1  shows a plan view of a component with a schematic indication of a channel with the individual areas; 
         FIG. 2  shows various embodiments and views of arrangements for separating particles, with the plan view of a first arrangement ( FIG. 2   a ), the corresponding sectional illustration in the center of the channel ( FIG. 2   b ), and the plan view of another electrode arrangement for separation ( FIG. 2   c ); 
         FIG. 3  shows the electrode arrangement for positioning the particles in the fluid flow in plan view ( FIG. 3   a ) and in a lateral sectional illustration ( FIG. 3   b ); 
         FIG. 4  shows the combination of an electrode arrangement according to  FIGS. 2   a  and  3   a  in plan view; 
         FIG. 5  shows two different electrode arrangements for the measuring channel area, once with a narrow measuring channel ( FIG. 5   a ) and with a wider channel ( FIG. 5   b ); 
         FIG. 6  shows various electrode arrangements for the alignment and sorting in the sorting area, having a first geometric arrangement ( FIG. 6   a ), a second, different geometric arrangement ( FIG. 6   b ), a third geometric arrangement of only the electrodes for leading away the particles sorted out ( FIG. 6   c ), and the arrangement according to  FIG. 6   a  in an enlarged illustration with the path of the particles ( FIG. 6   d ). 
     
    
    
     DETAILED DESCRIPTION 
     The schematic view in  FIG. 1  shows a plan view of a microfluidic component  1 , which shows an elongate channel  2  which on the left-hand side has an inlet opening  3  and on the right-hand side has two outlet openings  4  and  5 . Of course, a corresponding component  1 , if necessary, can also have a plurality of outlet openings or else inlet openings. The inlet openings can be connected to appropriately suitable devices for feeding a fluid, which can be liquid or possibly also gaseous. Accordingly, the outlet openings  4  and  5  are also connected to appropriate devices to accommodate the particles sorted out. In the following text, in connection with the exemplary embodiments, a liquid will be assumed, which transports various types of particle, for example biological cells. 
     The channel  2  is divided into a preparation area  6 , a measuring channel area  7  and a sorting area  8 . The cross sections of the individual areas are different, in particular the measuring channel area  7  being substantially narrower than the two other areas  6 ,  8 . In the individual areas  6  to  8  there are schematically indicated electrode arrangements  9  to  11 , which can be connected via associated conductor tracks  12  to an external control and measurement device, not illustrated. The individual areas with their electrode arrangements are explained further in the following text in connection with the figures. 
     The production of such a component can be carried out, for example, in that, on a glass substrate, first of all a lift-off resist for defining the positions of the electrodes is applied photolithographically. Following the application of an adhesive layer, for example titanium, a noble metal, for example platinum, as electrode material and a conductor material, for example copper, for a connection of the electrodes, are then deposited. Following the removal of the lift-off resist, polyimide is applied photolithographically as channel walls. The substrate is then joined together with an identical second substrate to form a sandwich structure with enclosed channels and electrodes. The thickness of the platinum layer can in this case be about 200 nm, and the thickness of the titanium layer can be about 50 nm. The polyimide layer is 1 to 20 μm. 
     It is also possible to use a different material, for example plastic, as the substrate material, and to use replication methods. 
       FIG. 2  shows various ways of separating particles.  FIG. 2   a  depicts the plan view of an electrode arrangement having a first separation electrode  13  and second separation electrodes  14 ′ and  14 ″. The two separation electrodes are arranged obliquely with respect to the walls  15 ,  16  and, as a result, have a funnel-like shape, which has the effect that the particles  17  or particle clusters  18  moving in this channel area are moved on the basis of the field forces. 
     In the plan views of the following Figures, as a rule only the upper electrodes are shown. As mentioned previously, in order to influence the particles on the basis of dielectrophoresis, it is obvious that there is also a corresponding lower electrode, which interacts with the upper electrode and therefore builds up the corresponding electric field which has the effect of influencing the particles. 
     The separation electrodes  13  can of course also be constructed with oblique electrode parts tapering to a point. The electrodes  14 ′,  14 ″ are arranged obliquely with respect to the walls  15 ,  16 , substantially parallel to the first separation electrode  13 . As distinct from the separation electrode  13 , they do not touch each other at the end, but leave an opening  19  for the passage of the particles  17 . The opening  19  corresponds to the average particle diameter or is slightly larger. The separation electrodes  14 ′ and  14 ″ are connected permanently and therefore constitute a barrier which holds up the particle clusters  18 . The electrode  13  is pulsed and therefore breaks the cluster up. This is shown by way of example in the side illustration according to  FIG. 2   b . As soon as the particles  17  have been detached from the particle cluster  18 , they can pass through the opening  19 . 
       FIG. 2   c  shows another variant having a different configuration with a plurality of separation electrodes  13  and  14  arranged beside one another. The distance between the separation electrodes  13  and the separation electrodes  14  in all the embodiments is chosen such that it is greater than the particle diameter, but smaller than the particle cluster size to be expected. 
     For the subsequent alignment of the particles  17  in a path at a desired position in the channel  2 , an electrode arrangement according to  FIG. 3   a  can be used. This figure shows the plan view of funnel-like positioning electrodes  20  and  21  each having an electrode arm  22  running obliquely with respect to the wall  15  and  16  and parallel electrode arms  23 . All these electrodes are switched on, the positioning electrode arms  22  having the effect that particles  17  moving in the channel move laterally toward the center. The positioning electrode arms  23  effect the vertical positioning of the particles, as shown in  FIG. 3   b . It is important here that the length of the parallel positioning electrode arms  23 , opposite which, as mentioned above, there are corresponding electrodes on the underside of the channel, is long enough to permit the vertical forces acting on the particles to find an equilibrium state, so that the height of the outlet path of the particles  17  is constant and, in this case, is centered in the channel. As a result of the lower electrodes, there is a total of four electrodes and, since the force on the particles  17  is higher when they approach the electrodes, in the case of a symmetrical electrode design, said electrodes tend to be centered at the center. The spacing of the electrodes should therefore also be chosen such that the particles are influenced by both electric fields (field from the electrode arms  22  and field from the electrode arms  23 ). The spacing between the positioning electrode arms  22  and  23  is only slightly greater than the particle diameter. 
       FIG. 4  shows an electrode arrangement from the combination of electrodes according to  FIG. 3   a  and  FIG. 2   a , the pulsed separation electrode  13  in this exemplary embodiment being configured so as to taper to a point. The spacing of the oblique electrode parts is two to four times the particle diameter. 
       FIG. 5  shows an exemplary embodiment of the channel  2  in the measuring channel area  7 , with an appropriate electrode arrangement  10 . The electrode arrangement  10  in this exemplary embodiment comprises, in a known manner, a first electrode pair  24  and a second electrode pair  25 , wherein each pair build up an electric field. The liquid and the particles flow through the channel  2  of the measuring channel area  7 . For most cases, a liquid contains an electrolyte of an impedance different from the particles. The first  24  and second  25  electrode pairs are connected to an electrical system, not illustrated, which measures the voltage across the individual electrodes and the current through the areas which are formed by the pairs of measuring electrodes  24  and  25 . Due to the difference between the impedance of the electrolyte and that of a particle, the measurement and control devices can ascertain when a particle passes through the first  24  or second  25  electrode pair fields. Each time a particle passes through the area of the first measuring electrode pair  24 , the aforementioned electrical parameters change. The area of the second measuring electrode pair  25  remains unchanged and serves as a reference. The behavior is appropriately reversed when a particle  17  passes through the second measuring electrode pair  25 . Using the first  24  and second  25  electrode pair impedance measurements, the velocity of a particle in the measuring channel area  7  may be obtained. 
     By way of background, velocity is the rate of change of displacement from a fixed point The average velocity v of a particle moving a distance d in a straight line during a time interval t is described by 
     
       
         
           
             
               
                 
                   v 
                   = 
                   
                     
                       d 
                       t 
                     
                     . 
                   
                 
               
               
                 
                   ( 
                   1 
                   ) 
                 
               
             
           
         
       
     
     The distance d between the first  24  and second  25  electrode pairs in the measuring channel area  7  is known and the time t a particle passes from the first electrode pair  24  to the second electrode pair  25  may be derived by the measurement and control devices. The measurement and control devices may therefore yield velocity. 
     The change in the electrical parameters is measured at the same time at a plurality of alternating current frequencies, which permits better differentiation and more accurate determinations of the parameters. The measured parameters are used by a control and measuring device, not illustrated, to carry out the subsequent sorting. 
       FIG. 5   b  shows a different configuration having a wider channel  2  in the measuring channel area  7 , a pair of deflection electrodes  26  deflecting the incoming particles  17  to a specific particle path, which leads through the measuring electrode pair  24 . The measuring electrode pair  25  is arranged in the area of the channel in which there are no particles  17 . The measuring electrode pair  25  therefore always serves as a reference. This configuration has the advantage that the risk of blockage is reduced by the greater channel width. Nevertheless, the measurement can be carried out by means of correspondingly small configurations of the electrodes as in  FIG. 5   a , in spite of an enlarged channel width. The electric fields are therefore concentrated in relation to the particle sizes, that is to say the influence of a large particle on the field is greater than the influence of a smaller particle. The measurements therefore become more accurate if the particle covers the greatest possible area of the field. The associated measurement electronics are neither shown nor further explained here (in this regard, see the aforementioned article by S. Gawad). 
       FIG. 6  shows various electrode arrangements  11  in the sorting area  8 . This electrode arrangement permits fast sorting of the particles.  FIG. 6   a  shows two alignment electrodes  27 ,  28 , which bring the particles  17  coming from the measuring channel in the exemplary embodiment into an area in the vicinity of one wall  15  of the channel  2 . Both alignment electrodes  27 ,  28  are switched on. The electrode  29  is the actual sorting electrode which, depending on whether a particle  17  is to remain on the path predefined by the alignment electrodes  27  and  28 , is switched off or else, if the particle  17  is to be deflected, is switched on. The deflection electrode  30  carries the deflected particles away and, owing to an opening  31 , permits the particle to emerge, so that division of the particles  17  in the direction of the two outlet openings  4  and  5  according to  FIG. 1  is possible by means of the following fork  32 .  FIG. 6   b  shows another alternative arrangement of the alignment electrodes  27  and  28 , the electrode  27  being designed to be shorter than the following sorting electrodes  29  and serving to carry the particles  17  away from the wall  15 . The deflection electrode  30  exhibits a different geometric arrangement here, as an example, without influencing the output result. In  FIGS. 6   a  and  6   b , in the case of the deflection electrode, both the electrodes, that is to say the upper and lower electrode, are illustrated in parallel, in order to illustrate the fact that the desired guidance of a particle  17  takes place only in the area in which the lower and the upper deflection electrode are illustrated as parallel. In the remaining area, the opening  31  for the passage is produced. 
       FIG. 6   c  shows a further possible configuration of the deflection electrode pair  30 . 
     The sorting operation will be explained in more detail by using  FIG. 6   d . It shows two possible paths for the particles  17 . As already mentioned, the alignment electrodes  27 ,  28  and the deflection electrodes  30  are always switched on. The sorting electrode pair  29  is activated when a particle is to be sorted out. This has the effect that the particle is deflected out of the intrinsically provided particle path  33  and is moved along the particle path  34  by the fluid flow. The deflection electrodes  30  do not overlap on the same section, so that, as a result, they form an opening  31  for the particles, which can then move onwards on a path substantially parallel to the particle path  33 . In order to achieve fast sorting, it is important for the length of the sorting electrode  29  to be as short as possible but long enough that, when it is switched on, the particle  17  continues its path toward the electrode  30  and the action of switching off the sorting electrode pair  29  for the next particle can be carried out as soon as possible. The deflection electrode  30  is arranged in such a way that particles which are not sorted out by the sorting electrode  29  can continue their path without being influenced too greatly by the deflection electrode  30 . 
     For the processes controlled by means of dielectrophoresis, voltages in the region of 10 V and frequencies from 100 kHz to 10 MHz are used, depending on the liquid and the particles. For the measurements in the measuring channel, the voltage is less than 2 V and the frequencies are 10 kHz to 200 MHz. 
     With the microfluidic component described above, integrated, fast and cost-effective “on-chip” detection with subsequent division is possible.