Abstract:
A microfluidic analytical apparatus ( 10 ) comprising a microfluidic card pairing ( 12 ) with a corresponding circuit card ( 14 ) and the circuit card pairing ( 12 ) with corresponding conductive fingers ( 16 ). The microfluidic card ( 12 ) has a plurality of channels ( 32 ) and ports ( 54 ) on a top surface of the card in a desired configuration, the ports ( 54 ) in fluid communication with the channels ( 32 ). The circuit card ( 14 ) has a plurality of conductive pins ( 60 ) projecting from a bottom surface of the card and having a configuration that corresponds to the particular configuration of the ports ( 54 ) of the microfluidic card ( 12 ) that is paired with. The circuit card ( 14 ) is received within a holder ( 20 ) that provides that provides multiple functions. Conductive pads ( 18 ) are disposed on a top surface of the circuit card ( 14 ), the pads ( 18 ) in electrical communication with the conductive pins ( 60 ) and corresponding to a configuration of conductive fingers ( 16 ) connected to a power source to provide voltage to the microfluidic card ( 12 ) for analytical operations. Additionally, detection difficulties associates with non-uniformities present on the card surface are overcome using a detection system incorporating a compliantly mounted microscopic head.

Description:
This invention relates in general to microfluidic devices and analytical apparatus for using microfluidic devices to conduct chemical and biochemical sample analysis. 
     BACKGROUND OF THE INVENTION 
     Today&#39;s microfluidic chips are capable of reliably carrying out many chemical and reactions and analytical assays using minimal amounts reagents. These high throughput cards incorporate arrays of fluidic networks, each network having a multitude of ports or reservoirs and microchannels associated therewith. Examples of microfluidic chips, fluidic arrays, and their methods use are described in U.S. Pat. Nos. 5,750,015; 6,103,199 and published patent application Ser. No. 99/19717 assigned to the assignee and hereby incorporated by reference. In each network, reservoirs are provided for introduction of sample, reagents, test compounds, or liquid media. In some cases, microfluidic devices are manufactured with media already in the channels or reservoirs as appropriate. 
     Microfluidic chips have been used for separation and analysis of nucleic acids, proteins and other molecules. By utilizing electrokinetic methods such capillary electrophoresis (CE), dielectrophoresis, and isoelectric focusing, components of a sample can be resolved and analyzed. One method of species detection involves conventional laser induced fluorescence, also known as LIF. A variety of mechanisms known in the art can be used for this purpose. For example, fluorescent detection mechanisms can be used in conjunction with confocal microscopy. Publications such as U.S. Pat. No. 5,296,703 and PCT WO 98/49543 describe systems for detecting fluorescent signals in microchannel arrays. 
     Desirably, microfluidic chips can be manufactured from a variety of polymer materials leading to user convenience, disposability and affordability. These materials allow for standard manufacturing techniques including injection molding, compression molding, casting or hot embossing. One drawback however is that these methods all require heating and cooling of the chip substrate. Given variations between substrates in glass transition temperatures and varying exposure to both ambient and elevated temperatures, the resulting chips often include some level of warpage and/or minor defects. These irregularities can interfere with the intended operation of the chip. For example, detection systems may include robotics programmed to move to specific locations on a planar card. If the card is warped, these locations are difficult to reach or become inaccessible. Accordingly, it is desired to provide an accurate detection system that can compensate for inherent deficiencies in the microfluidic chip, such as warpage. 
     Another issue arises due to the fact that the intended application of a microfluidic chip generally dictates its design. For instance, longer CE separation channels are required for sequencing of long nucleic acid sequences while smaller and more concise CE networks can be used to conduct multiplexed enzyme assays. The result is that for different applications, the layout of fluidic network arrays from chip to chip will be different. Conventional analytical systems incorporate circuit (electrode) cards and voltage sources as fixtures. Accordingly, their versatility is limited, usually resulting in expensive systems dedicated to particular applications. For this reason, it is desired to have an analysis and detection system with the versatility to accommodate different chip designs in multiple configurations. Additionally, the chemical and biochemical reactions carried out in microfluidic chips are conducted using small quantities of sample and other fluids that easily evaporate. Therefore, a need also exists for a microfluidic analytical apparatus that alleviates evaporation of fluids within microfluidic chips. 
     SUMMARY OF THE INVENTION 
     The above mentioned objects are achieved with a microfluidic analytical apparatus featuring a microfluidic chip having a configuration of ports in connection with channels and a circuit card having a surface with an array of conductive pin groups aligning with and corresponding to the microfluidic ports, with pins terminating in conductive pads disposed on another surface of the circuit card, the conductive pads aligning and being in electrical communication with conductive fingers providing voltages. 
     In other words, the present invention pairs a microfluidic chip or card described above with a corresponding circuit card. The circuit card can be used repeatedly to provide voltages to microfluidic cards having the corresponding configuration in electrokinetic operations such as electrophoretic separation of analytes, the electrophoretic movement of molecules into or out of reaction chambers, isotachophoretic concentration of molecules, electroosmotic movement of fluidics through channels or chambers of the microfluidic card, or the like. The microfluidic card has a plurality of channels and ports on a top surface of the card, the ports in fluid communication with the channels. A multitude of configurations, including various numbers of channels and ports in various locations, are incorporated into different microfluidic cards. The circuit card has a plurality of conductive pins projecting from a bottom surface of the card and having a configuration that corresponds to a particular configuration of the ports of the microfluidic card with which it is paired. 
     The circuit card is received within a holder that provides multiple functions. In one embodiment, the holder acts as a stop, which results in the suspension of the pins within the corresponding ports when the circuit card is paired with the microfluidic card. Therefore, the conductive pins of the circuit card contact the fluid within the ports or electrical circuits within the card ports, but not the ports themselves. Additionally, when the conductive pins of the circuit card are received within the ports, the holder contacts the microfluidic card and provides a seal between the microfluidic card and the circuit card thus assisting in preventing evaporation of material within the ports. 
     An electrical connection between the microfluidic card and circuit card of the present invention is simple to form when the conductive pins, in electrical communication with a power source, are inserted within the corresponding ports. Conductive fingers connected to the power source provide voltages to the microfluidic card through the conductive pins of the circuit card. The pins of the circuit card are arranged in groups. The pins in each group are electrically connected through electrical traces to conductive pads that terminate on a top surface of the circuit card. The conductive pad configuration corresponds to the configuration of the conductive fingers. The conductive fingers contact the conductive pads and provide voltages to the pads, which travel to the traces and the conductive pins. When the conductive pins are received within the ports voltages are provided through the conductive fingers and various operations, including molecular separations of materials within the channels, can then take place. 
     During sample separation detection mechanisms known in the art are used for sample analysis. Detection is usually optical and usually the signal is generated by laser-induced fluorescence; the detector can be a confocal optical system known in the art. Other detection mechanisms, such as electrochemical detection, may also be employed In one embodiment of the invention, the detection mechanism such as a microscope is disposed within a holder that moves vertically during analysis in relation to the microfluidic card so as to maintain a constant distance from the surface of the microfluidic card. In one embodiment the microscope has a compliantly mounted head that is in sliding contact with the microfluidic card during analysis. The compliantly mounted head moves vertically in response to any non-uniformities or warpage that the card may have without requiring refocusing of the detection optics, since a constant distance from the optics to the card is maintained. This embodiment is particularly useful when microfluidic cards are made of plastic, or contain plastic components, such as covers, or the like, that although having well defined and precise small-scale structural features such as channel widths, wall thicknesses, port diameters, and the like, are susceptable to warpage, bends, and other defects, from manufacturing processes, handling, sample preparation, loading, or the like. 
     Support frames are provided for the circuit card and the microfluidic card. In one embodiment the support frames are adapted for movement of the cards in relation to the confocal microscope. 
     Apparatus according to the invention assist in providing multiple microfluidic manipulations at high throughput rates to allow for continuous processing of high number of analyses at high rates of speed. The complexity of mass screening programs is reduced for example by the simple to use configuration of the conductive fingers with respect to the conductive pads and the configuration of the conductive pins with respect to the microfluidic parts, thereby eliminating many of the manipulation steps that are required in the use of convention analytical apparatus. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is an exploded view of the apparatus of the present invention. 
         FIG. 2  is a perspective view of the apparatus of the present invention pictured in FIG. I in conjunction with support frames. 
         FIG. 3  is a perspective view of upper and lower frames of the apparatus of  FIG. 2  supporting a microfluidic card and a circuit card. 
         FIG. 4  is an exploded view of the circuit card and the microfluidic card of the present invention pictured in  FIG. 1 . 
         FIG. 5  is a top view of a bottom surface of the circuit card of  FIG. 4 . 
         FIG. 6  is a plan view of the microfluidic card of  FIG. 4  and of a detection mechanism 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     With reference to  FIGS. 1 and 2 , there is seen an embodiment  10  of the present invention featuring a microfluidic card  12  paired with a circuit card  14  and conductive fingers  16  paired with conductive pads  18  of the circuit card  14 . The conductive fingers  16  are electrically connected to and in electrical communication with a power source (not shown) and provide voltages to the circuit card  14  during analysis of sample within the microfluidic card  12 . A detection system is employed for analysis of sample materials. In this embodiment of the invention, a microscope  42  is used as a part of the detection system; however, various detection systems known in the art may be used. 
     A holder  20  having wings  22 ,  24  and  26  holds the circuit card  14 . Wing  26  can be used to grip the holder  20 . The holder  20  is made from a rigid material such as for example, a rigid plastic. 
     With reference to  FIGS. 2 and 3  it is seen that wings  22  and  24  of circuit card  14  slide within partially enclosed channels  28  and  30 , respectively of an upper support frame  38  and a distal end  25  slides within partially enclosed channel  32  of the upper frame. Grip  26  is proximal to the user and can be used to insert the holder within the channels. 
     Shelves  34  upon which the microfluidic card  12  rests are also seen. The shelves  34  are a part of a lower support frame  36 . Other ways of maintaining the microfluidic card in place include clips, channels, grooves, adhesives, vacuums and differential pressure. 
     When a downward force is applied to the upper frame  38  the holder  20  and the circuit card  14  held by the holder move in a downward direction so that the circuit card  14  makes contact with and pairs with microfluidic card  12  as seen in  FIG. 2  and as described with reference to  FIG. 4  below. A securing device such as a pair of clamps  40  and  41 , attached to the frames  36  and  38 , maintains the downward position of the upper frame  38  and thus the circuit card holder  20 . Each clamp includes for example, upper plates  43  and  45  having apertures (not shown) attached to the upper frame  38 , lower plates  47  and  49  having apertures  51  and  53 , and fasteners  55  and  57  insertable through the apertures to clamp upper frame  38  down upon lower frame  36 . When the clamps  40  are released (for example the fastener is removed from the apertures) the upper frame  38  is moved back to an upward position away from lower frame  36  as seen in  FIG. 3 . Alternatively, pneumatic, electromagnetic or electromechanical securing mechanisms, as well as other mechanisms known in the art, may be used in addition to or in replacement of clamps  40 . 
     Positioned beneath the lower frame  36  is the microscope  42  disposed within a holder  44  adjacent to the microfluidic card  12  (when card  12  rests upon shelves  34 ) used for detecting migrated samples within the card  12 . The microscope includes a lens  46 , which is facing the microfluidic chip  12 . The microscope holder  44  is attached to an air cylinder  48 , which provides vertical movement to the microscope  42  through the holder  44 . In one embodiment additional actuators provide lateral movement Other mechanisms for providing responsive vertical movement of the microscope optical head or lens known in the art may be used, such as springs or similar mechanical devices, electromagnetic suspension of the type used in optical readers of compact disc players, and the like. 
     With reference to  FIG. 4  there is seen the microfluidic card  12  used in conjunction with the present invention having an upper first opposed major surface  50  and a lower second opposed major surface  52 . The card  12  may include various configurations of ports  54  on the surface  50  and channels  56  within surfaces  50  and  52  and is not limited to the configuration shown here. The configuration of channels and ports are within a solid substrate making up the card, which may be an inflexible substrate or a flexible substrate, such as a film. If the card is flexible, it will usually be supported and oriented in conjunction with a rigid support. In the present example, the card  12  being used is non-flexible, therefore it is not used in conjunction with a rigid support. A portion of the microchannels  56  is used as a detection site, to detect migrated samples. The channels comprising the detection site will generally have a depth of about 10 to 200 μm and a width ranging from about 1 to 500 μL The channels may be parallel or in various arrays and configurations. Depending on the purpose of the chip and the pattern of the channels, whether the channels are straight, curved or tortuous, the chip may only be 1 or 2 cm long or 50 cm long, generally being from about 2 to 20 cm long. The width will vary with the number and pattern of channels, generally being at least about 1 cm, more usually at least about 2 cm and may be 50 cm wide. The chip has ports, usually reservoirs for materials such as sample, buffer and waste that are connected to the channels. Additional channels may be connected to the main channel for transferring samples and reagents, etc. to the main channel. 
     The circuit card  14  has upper and lower opposed major surfaces. With reference to  FIGS. 4 and 5  a lower second opposed major surface  58  of the circuit card  14  will now be described. A phantom view of the lower second opposed major surface  58  of the circuit card  14  is seen in  FIG. 4  and a top plan view of the second opposed major surface  58  is seen in  FIG. 5 .  FIGS. 4 and 5  show an array of conductive pins  60  protruding from the second major surface  58 . The pins  60  have a configuration that corresponds to a particular configuration of the ports  54  of the particular microfluidic card  12  that it is paired with. For instance, in this example the array of pins  60  has the same configuration as the ports  54  pictured on the microfluidic card  12  ( FIG. 4 ) and the number of pins  60  is equal to the number of ports  54 . The conductive pins  60  are fine wires used as electrodes mounted on the underside  58  of circuit card  14 . The wires are usually platinum or other material with good electrical conductivity, substantially nonreactive (for example, platinum and gold) and corrosion resistant having a diameter of for example, 200 to 500 micrometers. The pins may be rigid or compliant For example, they may be spring-loaded or accordion-like. The electrode pins  60  act as cathodes and anodes for the separation of sample within the channels  50  and provide other voltages to the microfluidic card  12  for various operations. Electrode pins  60  may form wet or dry contacts with microfluidic card  12 . 
     With reference to  FIGS. 1 and 2  the upper surface of circuit card  14  is seen. First opposed major surface  62  of circuit card  14  has a plurality of conductive traces  64  that are connected to the array of conductive pins  60  on the second major surface  58 . Traces  64  terminate in conductive pads  18  on the top surface  62  electrically connected to conductive pins  60 . 
     The conductive pins  60  are arranged in groups of pins on the second major opposed surface  58  wherein a particular group of pins is electrically connected to the same conductive pad  18  through traces  64 . Traces  64  may be present on either the first or second opposed major surfaces  58  and  62 , on both of the surfaces, (as indicated in  FIG. 2 ), or in between the surfaces. The conductive pads  18  are electrically connected to an electrical power source (not shown) through conductive fingers  16  ( FIG. 1 ). The power source is provided with controls to change the voltages at the conductive fingers  16  thus at the conductive pads  18  in physical contact with the fingers  16  and to the conductive pins  60  in electrical contact with the pads  18 . The voltages are provided in a pattern determined according to the sequence of electroflow manipulations to be carried out in the microfluidic card during analysis. Conductive fingers  16  are made from any material with good electrical conductivity. Conductive fingers  16  extend from apertures (not shown) within block  66  and are connected to a power source at one end through electrical wiring  68  extending from block  66 . Wiring  68  provides voltages to the conductive fingers  16 . 
     The conductive fingers  16  are arranged in a configuration that corresponds to the configuration of conductive pads  18  found on the first major surface  62  of circuit card  14 . In  FIGS. 1-3  eight conductive fingers  16  are seen arranged in columns  70  of two fingers  16 . Three of the columns  70  are grouped together and a space  78  separates them from a fourth column. Conductive pads  18  on circuit card  14  are arranged in the same configuration as the conductive fingers  16 . Specifically, the pads  18  are arranged in groups of three columns close together and a fourth column spaced apart. In the example pictured there are more conductive pads  18  than conductive fingers  16 . However, in another embodiment the same number of conductive pads as conductive fingers is present. Arranging the conductive pads  18  in the same configuration as the conductive fingers  16  provides that an electrical connection between conductive pads  18  and conductive fingers  7  . . .  6  is easily established as the conductive fingers  16  pair with the conductive pads  18 . The conductive fingers  16  move horizontally along the top surface  62  of the circuit card  14  and move vertically to align and make contact with conductive pads  18 . As the conductive fingers are moved along to make contact with pads  18  various groupings of pads  18  are provided with voltages. These voltages are provided to pins  60  and ports  54  of the microfluidic card  12  so that sample analysis may occur. 
     With reference to  FIGS. 2-3 , lower frame  36  and upper frame  38 , in one embodiment, can move in a lateral or vertical position along tracks (not shown) to properly position the microfluidic card  12  and circuit card  14  for analysis by the microscope  42 . 
     Referring back to  FIG. 4 , the conductive pins  60  are removably insertable within the ports  54  of the microfluidic chip  12 . As stated above with regard to  FIGS. 2 and 3  clamps  40  and  41 , attached to the frames  36  and  38 , maintain the downward position of the upper frame  38  supporting the circuit card holder  20 . The holder  20  rests against card  12  and the conductive pins  60  enter and are suspended within the ports  54 . In one embodiment, edges  80  of the holder  20  act as stops that prevent the conductive pins  60  from contacting a bottom surface of ports  54 . 
     Additionally, the clamps  40  and  41  press the holder  20  against the card  12  forming a seal over the card. Fluids are sealed in the ports  54  and channels  56  when this seal is formed inhibiting evaporation of the fluids contained within the ports and channels. 
     There are many possible configurations and numbers of ports  54  and channels  56  that can be present on microfluidic cards  12  and corresponding configurations and numbers of the conductive pins  60  that are present on the circuit card  14  dependent on the type of desired analysis. By manufacturing a circuit card  14  that has conductive pads  18  that are configured as the conductive fingers  16  are, it is easy to establish electrical connections to the conductive pads  18  and to the electrically connected conductive pins  60  thus providing voltages to the ports  54  and channels  56  of the microfluidic device  12  for a desired operation. Regardless of the configuration of ports  54  and channels  56  and corresponding conductive pins  60 , the configuration of the conductive pads  18  remains constant. The conductive pads  18  are configured to correspond to the arrangement of the conductive fingers  16 . Therefore, the conductive finger  16  configuration will not have to be altered before analysis takes place, increasing the efficiency of the analysis. Additionally, various types of microfluidic cards  12  and corresponding circuit cards  14  can be produced, and yet each card  12 , regardless of the configuration, can be easily interchangeable for use with the apparatus  10 . 
     When the conductive pins  60  are suspended within the ports  54  and are connected to the appropriate voltage sources through conductive pads  18  and conductive fingers  16 , samples from the ports  54  can be moved from the ports into the separation channels  56  using an electric field. The separation channels are loaded with an appropriate separation medium The voltages are changed to then separate the samples by means of electrophoresis. During sample separation a detection region on the microfluidic card  12  is scanned using microscope  42  pictured in  FIG. 1 . 
     With reference to  FIG. 6 , it is seen that in one embodiment of the present invention, the microscope  42  has a compliantly mounted optical head  82  within holder  44 . The head  82  includes at least one of the optical elements described below. The compliant mounting mechanism used to mount the head  82  can be any mechanism that provides a vertical movement to the head that is responsive to deformities, such as warpage, or other irregularities, in the surface of microfluidic card  12 . Preferably, the head is rigidly mounted in every direction except the vertical direction, i.e. the head is rigidly mounted in directions parallel to the surface of microfluidic card  12 . “Rigidly mounted” means that the xy-position above (or below) the surface of microfluidic card  12  is controlled by a user, e.g. through conventional position controller under computer program control, or the like. A wide variety of methods may be used to provide a compliant mounting for the lens that allows the lens to move vertically relative to the surface of the microfluidic card in response to deformities. Such methods include using a spacer mounted with the lens together with a forcing means for applying a force perpendicular to the surface of the microfluidic card onto the lens so that the spacer is held in slidable contact with the surface of the microfluidic card. Preferably, the spacer is a cylindrical spacer coaxially mounted with the lens so that optical signals&#39; emanating from a channel in the microfluidic card can be collected by the lens. Forcing means include springs, electromagnetic force, hydraulic force, such as compressed air, elastomeric materials, or the like. The head may move vertically through the use of, for example, air cylinder  48 . Air cylinder  48  is connected through a mechanical connection, for example, push rod  90  to head  82 . The air cylinder  48  provides vertical movement of the holder  44  along surfaces of the microfluidic card  12  including deformities  84 . Deformities  84  include non-planar or warped surfaces. Roller bearings  86  rigidly mount the head  82  in the lateral direction and prevent the head from pivoting with the holder  44 . The air cylinder  48  moderates the force that is applied to the head  82 . The air cylinder is, for example, a soft air cylinder providing for example, 1-2 pounds of force that pushes a piston (not shown) of the cylinder upwardly and downwardly in a spring-like manner. Therefore, the moderate force that the air cylinder  48  provides helps to prevent damage to the microscope head  82  and to the card  12  when the head  82  is moved vertically by the air cylinder  48  to contact the card  12 . As the head  82  is compliantly mounted it is able to move within the holder and to make contact with the card  12 . In one example, nose piece  88  of the head  82  is able to travel along in a sliding contact relation with a surface of the microfluidic card  12 , such as second opposed major surface  52  even when the card has deformities  84 , without damaging the head  82 . Nose piece  88  rests against the surface  52  of the card  12  and in conjunction with the air cylinder  48  and compliant mechanism allowing for vertical movement maintain lens  46  at the correct distance from the channels within the card. Therefore, a significant attribute of the microscope  42  is that it is able to track the samples within channels  56  through wall  55  even though the channels and sample may be located within a card having non-uniform surfaces, without requiring refocusing of detection optics. Samples within channels  56  that otherwise could not be detected without manipulation of the detection optics or accurately detected with prior art mechanisms relying on a uniform shape of card can now be detected with ease. 
     In another embodiment, optical head  82  of microscope  42  may be rigidly mounted with collar, or nose piece,  88  and lens  46  compliantly mounted within optical head  82  so that they are responsive to deformities, warpage, or other irregularities in the surface of microfluidic card  12 . 
     Optical elements within head  82  are elements known in the art used for sample detection. For example, these elements include an illumination beam  100  from illumination source  102  that passes through a lens  110 , which serves to collect divergent light The beam  100  is then reflected by dichroic mirror  112 , which reflects light of the excitation wavelength of interest to pass through the mirror. The reflected beam  114  is focused by lens  46  and forms a small sharp beam, which passes into the detection regions of channels  56 . Fluorophores within the channel will be excited and will emit light, which will exit the channel and be collected by lens  46 . The emitted beam  118  will pass through dichroic mirror  112  and through lens  120  which focuses light beam  118  on photodetector  122 . The photodetector converts this light to electric signals for processing. The method by which the microscope  42  uses the excitation beam to scan the microfluidic card  12  can be a conventional confocal optical system known in the art or other detection mechanisms known in the art may be employed. The arrangement of optical elements described above may be substituted with other arrangements and types of optical elements used for sample detection.