Patent Publication Number: US-6986837-B2

Title: Microfluidic controller and detector system with self-calibration

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application is a Divisional of U.S. patent application Ser. No. 09/374,878, filed on Aug. 13, 1999, now issued as U.S. Pat. No. 6,498,497 B1, which is hereby incorporated by reference for all purposes. This application also claims priority to U.S. Provisional Patent Application No. 60/104,260, filed on Oct. 14, 1998, now abandoned, which is hereby incorporated by reference for all purposes. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to a controller and detector system for microfluidic systems, and more particularly, to a microfluidic controller and detector system for use with assay systems for performing chemical and biochemical analyses. 
     Analysis of chemical and biochemical samples often requires detection and identification of the constituent elements of the sample. Microfluidic devices are often used to separate and control movement of the elements of the sample to detect a property of the elements with a detection system. Microfluidics technology moves small volumes of fluids through channels on a chip to perform a multitude of laboratory tests to obtain biochemical and chemical information. This laboratory-on-a-chip technology enables microfluidics systems to support a range of applications in drug discovery, bioanalytical research and medical diagnostics, including DNA, RNA, and cell analyses. 
     The microfluidic devices typically include multiple wells that are interconnected with microchannels for transport of the sample. Application of a voltage across the channels permits the electrophoretic migration of macromolecular species in the sample. The samples often include an intercalating dye that becomes more fluorescent upon binding to the species of the sample. The fluorescent dyes are used to identify and locate a variety of cell structures such as specific chromosomes within a DNA sequence. 
     A variety of devices have been designed to read fluorescent labeled samples. In general the devices include at least one light source emitting light at one or more excitation wavelengths and a detector for detecting one or more fluorescent wavelengths. The light source is often a laser that emits light at one narrow center wavelength (single mode laser). 
     Despite the improvements achieved using parallel screening methods and other technological advances, such as robotics and high throughput detection systems, current screening methods still have a number of associated problems. For example, screening large numbers of samples using existing parallel screening methods have high space requirements to accommodate the samples and equipment, e.g., robotics etc., high costs associated with that equipment, and high reagent requirements necessary for performing the assays. Additionally, in many cases, reaction volumes must be very small to account for the small amounts of the test compounds that are available. Such small volumes compound errors associated with fluid handling and measurement, e.g., due to evaporation, small dispensing errors, or the like. Additionally, fluid handling equipment and methods have typically been unable to handle these volume ranges within any acceptable level of accuracy due in part to surface tension effects in such small volumes. 
     What is desirable is an integrated system to increase productivity, increase time- and cost-efficiency, rendering conventional laboratory procedures less cumbersome, less labor-intensive and less expensive and requiring fewer highly trained personnel. 
     SUMMARY OF THE INVENTION 
     The present invention provides a microfluidic controller and detector system. The controller and detector system is typically configured to receive a fluidic chip including at least two intersecting channels. The system preferably includes a detection zone and a material direction system comprising an interface configured for contact with the at least two intersecting channels on a different side of an intersection formed by the at least two intersecting channels. The microfluidic controller and detector optionally further includes an optics block comprising an objective lens and is located within the housing adjacent the detection zone. Finally, the microfluidic controller and detector typically includes a control system coupled to the microfluidic controller and detector with a communication channel for controlling operation of the microfluidic controller and detector. The control system is configured for receiving and analyzing data from the optics block. 
     The microfluidic controller and detector system generally comprises a fluidic chip that includes at least two intersecting channels and a detection zone, a material direction system comprising an interface configured for contact with the at least two intersecting channels, an optics block having an objective lens disposed adjacent the detection zone, and a control system coupled to the optics block and adapted to receive and analyze data from the optics block. The interface may be an electrical interface and/or a vacuum port adapted for interface with a vacuum pump. 
     In one embodiment, the electrical interface optionally comprises at least three electrodes, each configured for electrical contact with one of the intersecting channels on a different side of an intersection formed by the intersecting channels. In another embodiment, the material direction system includes a lid connected to the electrodes such that when the lid is in a closed position, the electrodes are in electrical contact with the intersecting channels. In yet another embodiment, the electrical interface also includes a reference voltage source for calibrating the channel electrodes. In yet another embodiment, the interface to the fluidic chip includes a vacuum port for moving a material, such as fluids and/or charged chemical species, using vacuum or pressure. 
     Preferably, the optics block includes a light detector to detect light emitting from the detection zone via the objective lens. The light detector is typically selected from photodiode, avalanche photodiode, photomultiplier tube, diode array, imaging systems, and charged coupled devices. In one embodiment, the light detector is in communication with the control system. The optics block optionally further includes a detector lens assembly positioned adjacent the light detector through which light from the detection zone travels. In addition, the optics block optionally includes a light source operable to direct light toward the detection zone via the objective lens and a mirror that reflects light produced by the light source and transmits light emitted from the detection zone via the objective lens. The light source is typically a laser, a laser diode, or a light emitting diode. 
     In another embodiment, the microfluidic controller and detector system includes a mounting apparatus for focusing light from the light source onto the detection zone via the objective lens. The mounting apparatus preferably comprises a first and a second adjacent plate, a pivot, and an actuator for displacing the first plate relative to the second plate about the pivot. The mounting apparatus typically includes two actuators each for displacing the first plate relative to the second plate in a different direction about the pivot. The actuator preferably is a stepper motor coupled to a coupler, the coupler being coupled to the first plate and in movable contact with the second plate. In one embodiment, the coupler defines threads therearound and the first plate defines an orifice therethrough, the orifice having internal threads configured to engage the threads of the coupler. Preferably, the second plate includes a hard seat adapted to be in contact with the coupler. 
     According to another embodiment, a method of calibrating a plurality of electrical source channels generally comprises generating a first electrical reference input at a reference channel and a first electrical source input at each of the electrical source channels, measuring a first electrical value at each of the reference and electrical source channels, generating a second electrical reference input at the reference channel and a second electrical source input at each of the electrical source channels, the second electrical reference input and the second electrical source input being different from the first electrical reference input and the first electrical source input, respectively, measuring a second electrical value at each of the reference and electrical source channels, and determining a readout calibration factor as a function of a ratio of differences between the first measured reference value and the first measured source value and between the second measured reference value and the second measured source value. 
     The above is a brief description of some features and advantages of the present invention. Other features, advantages, and embodiments of the invention will be apparent to those skilled in the art from the following description, drawings, and claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The present invention will be readily understood by the following detailed description in conjunction with the accompanying drawings, wherein like reference numerals designate like structural elements, and in which: 
         FIGS. 1A and 1B  are a perspective view and an exploded perspective view, respectively, of an assembly of a microfluidic controller and detector system in accordance with the present invention; 
         FIGS. 2A and 2B  are a perspective view and an exploded perspective view, respectively, of a base plate assembly for a clam shell unit of the controller and detector system of  FIGS. 1A and 1B ; 
         FIGS. 3A ,  3 B, and  3 C are a top perspective view, a bottom perspective view, and an exploded bottom perspective view, respectively, of an electrode assembly for the clam shell unit of the controller and detector system of  FIGS. 1A and 1B ; 
         FIG. 3D  is a perspective view of another assembly of a microfluidic controller and detector system in accordance with the present invention; 
         FIGS. 4A ,  4 B, and  4 C are a bottom view, a side cross-sectional view taken at line  4 B— 4 B in  FIG. 4A , and an exploded perspective view, respectively, of an optic block assembly for the microfluidic controller and detector system of  FIGS. 1A and 1B ; 
         FIG. 4D  is a schematic of an optics detector circuit; 
         FIG. 5A  is an exploded perspective view of a kinematic mounting assembly of the microfluidic controller and detector system illustrated in  FIGS. 1A and 1B ; 
         FIG. 5B  is a simplified partial cross-section view of coupling of a stepper motor to plates of kinematic mounting assembly of  FIG. 5A ; 
         FIG. 6A  is a perspective view of a reader assembly of the microfluidic controller and detector system illustrated in  FIGS. 1A and 1B ; 
         FIG. 6B  is an exploded perspective view of the wiggler and reader assemblies of the microfluidic controller and detector system illustrated in  FIGS. 1A and 1B ; 
         FIG. 6C  is an exploded perspective view of the kinematic mounting assembly; 
         FIG. 7  is an exploded perspective view of a chassis assembly of the microfluidic controller and detector system illustrated in  FIGS. 1A and 1B ; 
         FIG. 8  is a schematic illustration of a microfluidic chip for use with the microfluidic controller and detector system illustrated in  FIGS. 1A and 1B ; 
         FIG. 9  is a schematic of an embodiment of a system control circuitry board; 
         FIG. 10  is a schematic of the reference high voltage channel control circuitry board  195  for calibrating an electrical source channel; 
         FIG. 11  is a schematic of a control circuitry board for each high voltage source channels; 
         FIG. 12  is a schematic of a control circuit for a high voltage board; 
         FIG. 13  is a simplified schematic illustrating one embodiment of circuitry for a high voltage control PCB assembly of a reference channel and various high voltage electrode channels for use with the microfluidic controller and detector system illustrated in  FIGS. 1A and 1B ; and 
         FIG. 14  is a simplified schematic of circuitry for a high voltage loop for use as the reference channel or one of the high voltage electrode channels in the microfluidic controller and detector system illustrated in  FIGS. 1A and 1B . 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     A microfluidic controller and detector with self-calibration are disclosed. The following description is presented to enable any person skilled in the art to make and use the invention. Descriptions of specific embodiments and applications are provided only as examples and various modifications will be readily apparent to those skilled in the art. The general principles defined herein can be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, the present invention is to be accorded the widest scope encompassing numerous alternatives, modifications and equivalents consistent with the principles and features disclosed herein. For purpose of clarity, details relating to technical material that is known in the technical fields related to the invention have not been described in detail so as not to unnecessarily obscure the present invention. 
       FIGS. 1A and 1B  are a perspective view and an exploded perspective view, respectively, of an assembly of a microfluidic controller and detector system  20 . Microfluidic controller and detector system  20  includes a housing  21 , preferably including a first portion  21   a  and a second portion  21   b . Housing  21  generally encloses a main unit  22 . A lid  23  is optionally rotatively coupled to housing  21  for covering a clamshell unit  24  supported by main unit  22 . 
       FIGS. 2A and 2B  are a perspective view and an exploded perspective view, respectively, of a base plate assembly  30  for clam shell unit  24  of controller and detector system  20 . As shown, clamshell unit  24  preferably includes a base plate assembly  30 . Base plate assembly  30  generally includes a base plate  32 , a heat sink  33  and two connector plugs  34 ,  35 . As shown, heat sink  33  includes a bore  36  defined therein. 
       FIGS. 3A ,  3 B, and  3 C are a top perspective view, a bottom perspective view, and an exploded bottom perspective view, respectively, of an electrode assembly  31  of the clam shell unit  24  of the controller and detector system  20 . As shown, clamshell unit  24  preferably includes an electrode assembly  31 . Electrode assembly  31  typically includes a connector unit  40  that includes a connector plate  41  and a connector receptacle  42 . The connector plate  41  is coupled to connector unit  40  in any suitable manner and holds connector receptacle  42  in place therein. 
     Electrode assembly  31  of the clamshell unit  24  optionally further includes a lid  43  rotatively coupled to detector connector unit  40  in any suitable manner. An electrode printed circuit board (“PCB”)  44  having a plurality of electrodes  45  is typically disposed in lid  43 . Electrode PCB  44  can be coupled to lid  43  in any suitable manner. Optionally, PCB  44  comprises a plate of hydrophobic material, such as KEL-F™, PCTFE, TEFLON™, polypropylene, polyethylene, on a side of PCB  44  shown in  FIG. 3B  which interfaces with the fluidic device such that electrodes  45  can be inserted therethrough. Electrodes  45  preferably extend to an opposing side of PCB  44  for connection to electrical leads (not shown). The plate of hydrophobic material, e.g., KEL-F™, PCTFE, TEFLON™, polypropylene, polyethylene, advantageously resists or reduces formation of condensation which could lead to electrical shorting. 
       FIG. 3D  is a perspective view of an alternative assembly of a microfluidic controller and detector system  20 ′. The microfluidic controller and detector system  20 ′ is similar to the microfluidic controller and detector system  20  described above. For purposes of clarity, only key differences between system  20 ′ and system  20  are noted below. 
     As shown, the microfluidic controller and detector system  20 ′ includes a housing  21 ′ and a lid  23 ′ rotatively coupled to the housing  21 ′ for covering a clamshell unit  24 ′. The clamshell unit  24 ′ typically includes a base plate assembly  30 ′ and an electrode assembly  31 ′. As shown, the clamshell unit  24 ′ does not include a lid, but rather, the electrode assembly  31 ′ of the clamshell unit  24 ′ is disposed on an interior side of the lid  23 ′ of the housing  21 ′. The electrode assembly  31 ′ includes a plurality of electrodes  45 ′ disposed therein for interfacing with a fluidic device, such as a microfluidic chip. In addition, the clamshell unit  24 ′ of the microfluidic controller and detector system  20 ′ provides a replaceable personality cassette. The electrode assembly  31 ′, or the personality cassette, is replaceable and is easily removed from the lid  23 ′ of the housing  21 ′ such that it does not require detaching the clamshell lid from the clamshell unit, as is typically the case with the above-described microfluidic controller and detector system  20  embodiment. For example, a given electrode assembly  31 ′ can be replaced with a differently configured electrode assembly  31 ′, if necessary, for a different type of chip. The electrode assembly  31 ′ is typically slidable into a track on the lid  23 ′ of the housing  21 ′. 
       FIGS. 4A ,  4 B, and  4 C are a bottom view, a side cross-sectional view taken at line  4 B— 4 B in  FIG. 4A , and an exploded perspective view, respectively, of an optic block assembly  50  for microfluidic controller and detector system  20 . Optic block assembly  50  is preferably disposed within the main unit and under the clamshell unit. Optic block assembly  50  generally comprises an optic block housing  51  in which an objective  52  is disposed. Optic block housing  51  is typically enclosed on one side by a cover plate  54  and on another side by an optic PCB  56 .  FIG. 4D  is a schematic of one embodiment of the optics PCB  56 . Optic block assembly  50  preferably comprises one or more light sources, e.g., a first and a second light source  58   a ,  58   b . The light sources can optionally be any number of light sources that provide the appropriate wavelength of light, including lasers, laser diodes, light emitting diodes (LED), and the like. As shown, first light source  58   a  is mounted within optic block housing  51  via a light source or laser mount  62 . Light from first light source  58   a  is typically focused by a first lens tube assembly  60   a . At least a portion of the light passing through laser lens tube assembly  60   a  then passes through a band pass filter  64   a  mounted to a laser lens holder  64   b  and disposed within an opening  64   c  defined by optic block housing  51 . A first dichroic mirror  66   a  is preferably axially mounted by a mirror spring  68   a  at a 45 degree angle of incidence relative to the incoming light from first light source  58   a . Dichroic mirror  66   a  and mirror spring  68   a  are preferably disposed within an opening  70   a  defined by optic block housing  51 . Dichroic mirror  66   a  filters light by passing certain wavelengths while reflecting other wavelengths. For example, first dichroic mirror  66   a  typically filters the light emitted from light source  58   a  by reflecting only light with a wavelength less than approximately 670 nm. A portion of the light reflected by dichroic mirror  66   a  then passes through a second dichroic mirror  66   b  to objective  52 . Second dichroic mirror  66   b  is mounted to a mirror spring  68   b  within an opening  70   b  defined by optic block housing  51 . Second dichroic mirror  66   b  typically, for example, filters the light emitted from light source  58   a  by permitting only light with a wavelength above approximately 585 nm to pass therethrough. 
     The light from first light source  58   a  that passes through second dichroic mirror  66   b  is focused by objective  52  and impinges on, for example, a sample within microfluidic system  20 . Fluorescence is typically emitted from the sample back through objective  52 . Fluorescence at certain wavelengths is permitted to pass through second dichroic mirror  66   b , through first dichroic mirror  66   a , and is then focused by lens tube assembly  72   a  towards a first light detector PCB  74   a.    
     Light from second light source  58   b  is generally focused by a second lens tube assembly  60   b . A third dichroic mirror  66   c  is preferably axially mounted by a mirror spring  68   c  at a 45 degree angle of incidence relative to the incoming light from lens tube assembly  60   b . Dichroic mirror  66   c  and mirror spring  68   c  are preferably disposed within an opening  70   c  defined by optic block housing  51 . Third dichroic mirror  66   c  can, for example, further filter the light emitted from light source  58   b  by reflecting only light with a wavelength less than approximately 505 nm. At least a portion of the light reflected by third dichroic mirror  66   c  is then reflected by second dichroic mirror  66   b  to objective  52 . Second dichroic mirror  66   b  can, for example, filter the light emitted from light source  58   a  by reflecting light with a wavelength less than approximately 585 nm. 
     The light from second light source  58   b  reflected by second dichroic mirror  66   b  is focused by objective  52  and impinges on, for example, a sample within microfluidic system  20 . Fluorescence is typically emitted from the sample back through objective  52 . Fluorescence at certain wavelengths is reflected by second dichroic mirror  66   b  and is permitted to pass through third dichroic mirror  66   c . The fluorescence passing through third dichroic mirror  66   c  is then focused by lens tube assembly  72   b  towards a second light detector PCB  74   b.    
     Each of lens tube assemblies  72   a ,  72   b  preferably includes a detection filter which filters the signal emitted from the sample. Detection filters clean up light emitted from the sample by removing scattered light such that light from the fluorescence light signal pass through while light from light source is filtered out. Lens tube assemblies  72   a ,  72   b  are positioned adjacent to light detector PCB  74   a ,  74   b , respectively. 
     Each of light detectors  74   a ,  74   b  converts incoming light into electric signals. Detection system  20  is preferably coupled to the host computer  198  (shown in  FIG. 1A ) via a serial connection for transmitting detected light data to the computer for analysis, storage, and data manipulation. Light detectors  74   a ,  74   b  is optionally a photodiode, avalanche photodiode, photomultiplier tube, diode array, or imaging systems, such as charged coupled devices (CCDs), and the like. Light detectors  74   a ,  74   b  optionally includes, for example, an integrator and an analog-to-digital converter having an analog input coupled to an output of the integrator, as described in U.S. patent application Ser. No. 09/104,813, filed Jun. 25, 1998 which is incorporated herein by reference in its entirety. 
     In one preferred embodiment, first light source  58   a  comprises a red laser or a red laser diode. The red laser or red laser diode facilitates detection of fluorescent species that excite in the red range. Second light source  58   b  preferably comprises a blue light emitting diode (“LED”) which can be used for multi-wavelength detection schemes and/or in less sensitive analyses, for example. First light detector  74   a  is preferably a photo diode where the lens tube assembly  72   a  includes a filter  76   a  for passing 682 nm centered wavelength with a bandwidth of approximately 20 nm. Second light detector  74   b  is preferably a photo diode where the lens tube assembly  72   b  includes a filter  76   b  for passing 525 nm with a bandwidth of approximately 20 nm. As shown, the filters  76   a ,  76   b  are contained in the lens tube assemblies  72   a ,  72   b.    
     Some aspects of some of the components and functionality of optic block assembly  50  is further described in co-pending U.S. Provisional Application No. 60/143,399 filed on Jul. 12, 1999, now abandoned, the entirety of which is incorporated by reference herein. 
     Although system  20  described above is described for use with a microfluidic device containing a sample with a fluorescent label, it is to be understood that the system may be used to detect other types of labels including light absorbing labels and radioactive labels, for example. 
       FIG. 5A  is an exploded perspective view of a kinematic mounting assembly  80  of the microfluidic controller and detector system  20 . Kinematic mounting assembly  80  is optionally coupled to optics block assembly  50  to align and focus the optics block relative to the analysis channel in the chip. 
     Kinematic mounting assembly  80  generally comprises a first and a second stepper motor  81 ,  82  mounted to a first plate  83  via an L bracket  84 . First plate  83  is positioned adjacent a second plate  85  movable relative to first plate  83 . First and second plates  83 ,  85  are movably coupled, such as by springs  86 ,  87 ,  88  coupled between the first and second plates with any suitable attachment mechanism such as set screws or pins (not shown). Three springs are preferred although one spring is generally centrally provided between first and second plates  83 ,  85 . 
       FIG. 5B  is a simplified partial cross-section view of coupling of first stepper motor  81  to first and second plates  83 ,  85  of kinematic mounting assembly  80  via a first coupler  89 . Coupler  89  comprises a ball shaped or rounded end  90 , a threaded rod  91  extending from ball shaped end  90 , and an internal opening  92  defined in rod  91 . Threaded rod  91  is configured to engage with threads  93  of first plate  83  such that rod  91  is rotatable relative to first plate  83 . 
     Internal opening  92  of rod  91  is optionally configured to slidably mate or slip fit with a shaft  94  of first stepper motor  81  such that rotation of the first stepper motor shaft result in rotation of coupler  89 . For example, the internal rod opening and first stepper motor shaft have mating hexagonal cross-sectional shapes such that internal rod opening  92  defines a hex socket which shaft  94  of first stepper motor  81  serves as a mating hex key. Thus, as first stepper motor  81  rotates shaft  92 , causing coupler  89  to rotate within first plate  83 , coupler is translationally displaced in a Y direction to thereby increase or decrease a distance between first and second plates  83 ,  85 . Alternatively, a flexible shaft coupling can be used. 
     Second plate  85  preferably provides a hard seat or surface  95   a  having approximately a diameter approximately same, one-half, one-fourth, or any suitable portion of a diameter of ball shaped end  90 . Hard seat  95   a  generally comprises a material such as cubic zirconium such that wear from movement of ball shaped end  90  over hard seat  95   a  is minimized. Ball shaped end  90  preferably similarly comprises a hardened material such that its shape and size do not generally change over time due to wear. 
     Such an internally threaded bushing driven by a stepper motor with a ball or a ball shaped end riding on a seat is known in the art. Any other suitable coupling of the shaft of the stepper motor to the coupler can optionally be implemented. For example, a flexible elastomer shaft coupling utilizing a helical spring can be utilized as the coupler. 
     Although not shown, second stepper motor  82  optionally has a configuration similar to that of first stepper motor  81 . For example, second stepper motor  82  includes a shaft configured to slidably engage or slip fit with an internal opening of a second coupler. Further, the internal rod opening and second stepper motor shaft optionally have mating hexagonal cross-sectional shapes such that the internal rod opening defines a hex socket to which the shaft of second stepper motor  82  serves as a mating hex key. 
     The second coupler generally comprise a ball shaped or rounded end, a threaded rod extending from the ball shaped end, and the internal opening to which the shaft of second stepper motor  82  is typically engaged. The threaded rod is optionally configured to engage with internal threads of a member or an extension stationary relative to and/or coupled to second stepper motor  82 , first plate  83 , and/or mounting bracket  84 , for example. A spring is preferably provided along a Z direction to couple second stepper motor  82  to second plate  85 . For example, the Z direction spring is typically coupled via a pin or a set screw to the member or extension on one end and to second plate  85  on another of the Z direction spring. 
     A side surface of second plate  85  preferably provides a hard seat or surface  95   b  having approximately a diameter approximately same, one-half, one-fourth, or any suitable portion of a diameter of the ball shaped end of the second coupler. Hard seat  95   b  is generally similar in construct as hard seat  95   a  and serves a similar purpose of minimizing wear from movement of the ball shaped end of the second coupler over hard seat  95   b . The second coupler similarly generally comprises a hardened material such that its shape and size do not generally change over time due to wear. 
     The configuration of second stepper motor  82  is such that rotation of its shaft causes rotation of the second coupler within the internally threaded stationary member or extension. The second coupler is thus translationally displaced in a Z direction to thereby rotate second plate  85  relative to first plate  83  about a pivot. 
     Thread engagement between the couplers and first plate  83  effectively gears down the stepper motors to allow for accurate and precise relative positioning of first and second plates  83 ,  85 . The resolution of such positioning is typically determined and selected based upon the threads and parameters of each stepper motor. Resolution of approximately 0.8 μm of displacement or travel for each step of the stepper motor can be easily achieved. 
     Kinematic mounting assembly  80  preferably provides a pivot about which second plate  85  is moved relative to first plate  83  in each of the Y and Z directions. In the embodiment shown in  FIG. 5A , kinematic mounting assembly  80  provides two pivots, each comprising a ball  96   a ,  96   b  and a corresponding seat  97   a ,  97   b , respectively. Seats  97   a ,  97   b  are optionally provided by first plate  83 . 
     One of seats  97   a ,  97   b  is optionally a cone shaped recess configured to receive approximately one-half of a corresponding ball therein such that the corresponding ball can rotate within the recess. The ball and cone shaped recess combination generally serve as a pivot for movement of second plate  85  relative to first plate  83  such as in the Y direction with actuation of first stepper motor  81  and/or in the Z direction with actuation of second stepper motor  82 . The other of seats  97   a ,  97   b  is optionally a hard surface seat similar to seat  95   a  (shown in  FIG. 5B ) such that a corresponding ball can slidably move in an X-Z plane. The ball and hard surface seat combination generally serves as a third contact point, in addition to contacts points provided by ball  90  of coupler  89  and the pivot comprising the cone shaped recess and the corresponding ball, to define a plane. 
     Each of balls  96   a ,  96   b  is typically attached by any suitable attachment mechanism to second plate  85 . Alternatively, balls  96   a ,  96   b  are unattached to and disposed between first and second plates  83 ,  85  and are confined to between first and second plates  83 ,  85  via springs  86 ,  87 ,  88 . 
     Preferably, first and second plates  83 ,  85  are coupled to the optic block assembly such that the first plate  83  is stationary relative to the base plate assembly  30  and the second plate  85  is coupled to the optic block housing. Alternatively, the second plate  85  are coupled to the objective such that the objective can be moved and positioned over a distance of up to approximately 3 mm, for example, to scan and locate channels or a detection window of a microfluidic chip as will be described below and/or such that the objective can be focused by displacing the objective in a Z direction, such as up to approximately 0.5 mm, relative to the detection window of the microfluidic chip. 
     In one preferred embodiment, each of balls  96   a ,  96   b  has a diameter of approximately 6 mm such that the pivot comprising the cone shaped recess and the corresponding ball provides a clearance between first and second plates  83 ,  85  of approximately 3 mm. 
       FIG. 6A  is a perspective view of a reader assembly  189  and  FIGS. 6B and 6C  are exploded perspective views of the reader assembly  189  and the kinematic mounting assembly  80 , respectively. Reader assembly  189  comprises the clam shell unit  24 , the optics block assembly  50 , the kinematic mounting assembly  80 , and an assembly cover  188 . The first and second stepper motors  81 ,  82  of the kinematic mounting assembly  80  and the L bracket  84  to which the motors  81 ,  82  are mounted form a wiggler assembly  180 . As shown, the first and second stepper motors  81 ,  82  of the kinematic mounting assembly  80  utilize spindles. The clamshell unit  24  is positioned over wiggler assembly  180  and optic block assembly  50  such that objective  52  of optic block assembly  50  is in alignment with bore  36  defined within heat sink  33  of clamshell unit  24  (also shown in  FIG. 2B ). 
       FIG. 7  is an exploded perspective view of a chassis  190  of microfluidic controller and detector system  20 . Reader assembly  189 , a control PCB assembly  191 , a power supply  192  and a cooling fan  193  are typically coupled to chassis  190  in any suitable manner. A connector  194  provided connection via a communication channel  194   a  to a control system  198  such as a computer (shown in  FIG. 1A ). Two high voltage PCBs  195 ,  196  are optionally provided. A chassis cover  197  encloses chassis  190 . 
       FIG. 8  is a schematic illustration of a microfluidic chip  100  for use with microfluidic controller and detector system  20 ,  20 ′. Microfluidic device  100 , such as a microchip, is typically placed within clamshell unit  24  on base plate  32  (shown in  FIG. 2 ) during operation. Microfluidic device  100  generally comprises a plate  102  defining a plurality of integrated network of channels  104  therein and a plurality of reservoirs  106 – 136  in various fluid communication with channels  104 . Buffers, reagents, and/or samples to be analyzed are placed into one or more of reservoirs  106 – 136  for introduction into one or more of channels  104 . Preferably, reservoirs  130 ,  132 ,  134  are waste reservoirs and reservoir  136  is a buffer reservoir. The fluids are transported from their respective reservoirs, either separately or together with other reagents from other reservoirs into a main analysis channel  138  and along the main channel to the waste reservoir  132 , past a detection region (or window)  140 . 
     The microfluidic device  100  is typically positioned within microfluidic controller and detector systems  20 ,  20 ′ with its detection region or window  140  disposed in an optical path of the objective of the optic block such that the system is in sensory communication with detection region  140  of main analysis channel  138 . The objective is preferably positioned at an appropriate distance for activating the fluorescent indicator within the test sample. As the sample passes the detection region  140 , signals produced by the sample materials are detected by systems  20 ,  20 ′. 
     Detection window  140  is preferably transparent so that it is capable of transmitting an optical signal from main channel  138  over which it is disposed. Detection window  140  can merely be a region of a transparent cover layer, e.g., where the cover layer is glass or quartz, or a transparent polymer material, e.g., PMMA, polycarbonate, etc. Alternatively, where opaque substrates are used in manufacturing microfluidic device  100 , transparent detection windows fabricated from the above materials is separately manufactured into the device. 
     Microfluidic device  100  preferably includes at least two intersecting channels and optionally includes three or more intersecting channels disposed within plate  102 . Channel intersections can exist in a number of formats, including cross intersections, “T” intersections, or any number of other structures whereby two channels are in fluid communication. Microfluidic device  100  preferably has multiple sample introduction ports or reservoirs, for the parallel or serial introduction and analysis of multiple samples. Alternatively, microfluidic device  100  is coupled to a sample introduction port, e.g., a pipettor, which serially introduces multiple samples into the device for analysis. 
     The samples are typically transported along main analysis channel  138  and past detection window  140  by vacuum pressure and/or the application of electric fields such as with electrokinetic transport systems, for example. The electrokinetic transport system directs materials along the interconnected channels through the application of electrical fields to the material, thereby causing material movement through and among the channels, i.e., cations will move toward the negative electrode, while anions will move toward the positive electrode. 
     Such electrokinetic material transport and direction systems include those systems that rely upon the electrophoretic mobility of charged species within the electric field applied to the structure. Such systems are more particularly referred to as electrophoretic material transport systems. Other electrokinetic material direction and transport systems rely upon the electrostatic flow of fluid and material within a channel or chamber structure, which results from the application of an electric field across such structures. 
     In brief, when a fluid is placed into a channel which has surface bearing charged functional groups, e.g., hydroxyl groups in etched glass channels or glass microcapillaries, those groups can ionize. In the case of hydroxyl functional groups, this ionization, e.g., at neutral pH, results in the release of protons from the surface and into the fluid, creating a concentration of protons at or near the fluid/surface interface, or a positively charged sheath surrounding the bulk fluid in the channel. Application of a current and/or a voltage gradient across the length of the channel causes the proton sheath to move in the direction of the current or the voltage drop, i.e., toward the negative electrode. 
     Microfluidic device  100  described herein is useful in performing a variety of analyses, such as characterization operations on biological macromolecules, e.g., proteins and/or nucleic acids, screening assays, electrophoretic separation of macromolecules (e.g., nucleic acids, proteins) and medium or high throughput screening assays, e.g., in pharmaceutical discovery and diagnostics as disclosed in U.S. patent application Ser. No. 08/8456,754, filed Apr. 25, 1997 and Published International Application No. WO 98/00231 which are hereby incorporated by reference in their entireties. The controller and detector system  20  in which the microfluidic device can be used is useful for detecting fluorescence induced by the buffers and/or samples from exposure of laser radiation to generate chromatographic data, for example. It is to be understood that the microfluidic device used with detection systems  20 ,  20 ′ of the present invention may be different from those described herein without departing from the scope of the invention. 
     In operation, a separation buffer is typically first placed into, for example, buffer reservoir  136 , and allowed to wick into channels  104 , thereby filling the channels with the separation buffer. Samples that are to be analyzed are separately placed into one or more of reservoirs  106 – 128 . The separation buffer, already present in reservoir  136 , is typically also placed into reservoirs  130 ,  132  and  134 . Movement of materials through the channels of the chip is accomplished by applying appropriate electrical currents and/or voltages through the channels to drive electrokinetic movement of the materials. Currents and/or voltages are supplied via electrodes  45  (shown in  FIGS. 3B and 3C ). Each electrode generally corresponds to a reservoir such that, in the exemplary embodiment shown and described, there are sixteen electrodes corresponding to sixteen reservoirs. 
     Through the application of appropriate electric inputs, a first sample material is transported or electrokinetically transported from its reservoir, e.g., reservoir  106 , to and through a main injection intersection  142  for main channel  138 , via channels  140   a  and  140   b . In one embodiment, this can be accomplished by applying a current between reservoirs  106  and  134 . Low level pinching currents are typically applied at intersection  142  in order to prevent diffusion of the sample material at the intersection, e.g., by supplying a low level of current from reservoirs  132  and  136  toward reservoir  134  (see, e.g., WO 96/04547, incorporated in its entirety by reference herein). 
     After a short period of time, the application of current is switched such that material in intersection  142  is electrokinetically transported through main analysis channel  138 , e.g., by applying a current between reservoirs  136  and  132 . Typically, a slight current is applied after the injection to pull materials in channels  140   b  and  140   c  back from intersection  142 , to avoid leakage into main channel  138 . 
     While the first sample is transported through main channel  138 , a second sample to be analyzed is typically preloaded by transporting the second sample material from its reservoir, e.g., reservoir  108 , toward preload reservoir  130  through preload intersection  144 . This allows for only a very short transit time to move the sample material from its preloaded position to injection intersection  142 . Once analysis of the first sample is complete, the second sample material is typically transported across injection intersection  142  and injected through main analysis channel  138 , similar to the process described above. This process is preferably repeated for each sample loaded into chip  100 . The desired analysis operations are carried out in analysis channel  138 , such as electrophoretic separation and screening interactions. Although generally described as incorporating electrokinetic material transport system, it will be appreciated that other systems can optionally be employed in addition to, or in lieu of such an electrokinetic system. For example, a vacuum source or pump is optionally provided in main unit  22  with connection via clamshell  24 . 
     A number of the components that are used in conjunction with the present invention have been described in commonly owned, copending applications, including, e.g., U.S. application Ser. No. 09/165,704, filed Oct. 2, 1998, U.S. application Ser. No. 08/919,707, filed Aug. 29, 1997, and Published International Application No. 98/05424, each of which is incorporated herein by reference in its entirety. 
     As noted above, the interaction of the first and second components is typically accompanied by a detectable signal. Generally, monitoring of the signals produced by the sample materials at the detection window is achieved by placing a laser light source at an appropriate wavelength for activating the fluorescent indicator within the test system. Fluorescence is then detected using the lens assemblies in combination with the detector PCBs as described above with reference to optic assembly  50 . The signals are preferably monitored by objective  52  (shown in  FIGS. 4B and 4C ). These signals are viewed by the lens assemblies which transmit the signals to their corresponding detectors. The PCBs then transmit the signals to the computer. The computer can then be used to analyze the signals and create various outputs, such as graphs, tables and charts. Furthermore, computer  198  (shown in  FIG. 1A ) is typically used to control microfluidic system  20  or  20 ′. Commands are input, through appropriate input means such as a keyboard or a mouse, to the computer which then transmits commands to control PCB assembly  191 . 
     Thus, the present invention provides a microfluidic detector and controller system that works with a microfluidic chip which is optionally constructed of two similarly bonded planar glass substrates. Referring again to FIGS.  2  and  3 A– 3 C, the microfluidic chip is typically placed onto base plate  32  within a clamshell configuration that includes controlling electrodes  45  that mate with holes (not shown) provided in an upper substrate of microfluidic chip  100 . Closure of clamshell lid  43  places the mating array of electrodes  45  into contact with the various reservoirs and thus the fluids contained within microfluidic chip  100 . Electrical inputs are generally delivered via electrodes  45  to the various reservoirs and serve to direct material transport through the interconnected channels by vacuum pressure, electrophoretic and/or electrosmotic movement, for example. 
     The channel network is filled with a separation medium. Preferably the separation medium used is a low viscosity solution of polydimethacrylade-co-acrylic acid. The DNA is labeled with the intercalating fluorescent dye “Syto-66 Super ™” which is available from Molecular Probes. Nucleic acid fragments are separated as they travel through the separation or main analysis channel due to their differing electrophoretic mobilities. These fragments take up the dye within the separation medium. 
     The fluorescent intercalating dye, associated with the fragments, are typically detected by objective  52  with light emitted from light source  58   a  and reflected off of mirror  66   a  and passed through mirror  66   b , as shown in  FIGS. 4A–4C . Alternatively, second light source  58   b  can transmit a blue light though objective  52  via lens assembly  60   b  and mirrors  66   c ,  66   b . Light is transmitted back and detected by one of detectors  74   a ,  74   b . One or both of these light sources and/or other light sources which may be alternatively or additionally provided are optionally used to activate the dye that is associated with nucleic acids within the main analysis channel. 
       FIG. 9  is a schematic of an embodiment of a system control circuitry board  191 .  FIG. 10  is a schematic of the reference high voltage channel control circuitry board  195  for calibrating all electrical source channels.  FIG. 11  is a schematic of a control circuitry board  196  for each of the  16  high voltage source channels.  FIG. 12  is a schematic of a control circuit for a high voltage board. 
       FIG. 13  is a simplified schematic illustrating one embodiment of circuitry  200  for high voltage control PCB assembly of a reference channel  202  and various high voltage electrode channels  204 ,  206 ,  208 ,  210  for use with microfluidic controller and detector system  20  or  20 ′. Each high voltage electrode channel is connected via an electrode to a reservoir defined in the microfluidic chip. As described above, each electrode generally corresponds to a reservoir such that, in the exemplary embodiment shown and described, sixteen electrode channels are provided to correspond with the sixteen electrodes which in turn correspond to the sixteen reservoirs. The reference channel is an extra channel provided to enable calibration of the electrode channels. Although shown with four electrode channels, circuitry  200  may include any number of two or more electrode channels in addition to reference channel  202 . 
     Electronic circuits drift, whether due to aging, temperature and/or humidity changes, and/or other causes. Electronic drifts affect the performance of the electronic circuit. For example, for microfluidic controller and detector system  20  or  20 ′, it is highly desirable to tightly control the voltage or current applied to the reservoirs via the electrodes. Generally, electronic drifts that match, i.e. drift by a same ratio, for all electrode channels do not significantly degrade the performance of the electronic circuit. However, if the applied voltage or current to one reservoir increases by, for example, 1% while the applied voltage to another reservoir decreases by, for example, 1%, such electronic drift could lead to chemical cross-talk between the contents of different reservoirs. Further, it is generally difficult to provide high voltage resistors that are stable over time and temperature for the level of precision desired for the microfluidic controller and detector system. Such high voltage resistors are used in resistor voltage dividers for each high voltage channel to measure and set the voltage of the channels. 
     Thus, the reference channel is provided in the circuitry for high voltage control PCB assembly as an extra channel for use in calibration of the electrode channels. Preferably, a calibration scheme or process is executed prior to each test or run to analyze the microfluidic chip. Because the circuitry for the reference channel is utilized only once for each test or run, effects of aging on the reference channel circuitry is reduced as compared to the electrode channels. Further, although described in terms of microfluidic controller and detector system  20  or  20 ′, the provision of the reference channel and the calibration process is optionally utilized in any system to ensure that voltages and/or currents for a plurality of channels match. 
     As shown in  FIG. 13 , reference channel  202  generally comprises a high voltage generator  212  which receives a DAC set-point output  214  as input. Reference channel  202  further includes a voltage divider comprising serially coupled first and second high voltage resistors  218 ,  220 . The voltage divider is coupled in parallel to high voltage generator  212 . A voltage  222  is taken between two nodes of second high voltage resistor  220 . In addition, a current  224  is taken between a node  230  coupled to high voltage generator  212  and second high voltage resistor  220  and ground. Output of the reference channel OUT REF  or output of each electrode channels OUT 1 , OUT 2 , etc. is taken at node  228 . Reference channel  202  is coupled to each of electrode channels  204 ,  206 ,  208 ,  210  via a low leakage high voltage diode  226 . Each high voltage electrode channels  204 ,  206 ,  208 ,  210  is of generally identical construct as reference channel  202 , except that they have a voltage or current mode select signal  216  as inputs. 
       FIG. 14  is a simplified schematic showing the feedback loop circuitry for first channel  204  in greater detail. As noted above, the circuitry for the high voltage electrode channels and for reference channel  202  are of generally identical construct. As shown, high voltage generator  212  of first channel  204  generally includes an integrator  232 , a transformer with voltage doubler  234 , a diode  236 , and an amplifier  238  for converting a current to voltage. High voltage generator  212  is controlled by a feedback loop that regulates output based on DAC setpoint output  214  and voltage and current readings  222 ,  224 . Voltage reading  222  and current reading  224  are sampled by an analog-to-digital converter to generate a digital value representation of the actual voltage and current on the output  240 . 
     Amplifier  238  is operated in such a way that node  230  is at virtual ground. During operation, the electrode channels are optionally set in all the same mode or in different modes. Because reference channel  202  preferably operates only in voltage mode, a portion of the circuit, e.g., switch  216 , need not be provided. 
     During normal operation or analysis of samples in the microfluidic chip, reference channel  202  is shut off such that no significant current flows between the reference channel and each of the high voltage electrode channels so long as the voltage at each high voltage electrode channel is at a positive or 0 voltage. In contrast, during calibration, voltage at reference channel  202 , i.e., voltage at reference node  228 , is set to a positive voltage at least an amount of a voltage drop across diode  226  greater than voltage of one or more high voltage electrode channels such that current can flow to those one or more of the high voltage electrode channels. 
     The following is a description of an exemplary calibration process although any other suitable calibration processes can optionally be utilized and numerous modifications can be made to achieve similar calibration results. 
     First, reference channel 202 and all the electrode channels are shut off. The voltage and current V RefReadOffset , I RefReadOffset  of reference channel  202  are measured. The voltages and currents V ChNReadOffset , I ChNReadOffset  of each electrode channel N, where N ranges from 1 to the number of electrode channels, such as sixteen, are measured. 
     Next, voltage at node  240  of all electrode channels are set to a 1200V set point voltage or V 1.2kVSetPoint  and voltage at node  228  of reference channel  202  is set to a 1000V set point voltage or V 1kvRefSetPoint . Because the actual 1000 V reference channel set point voltage may not be exactly equal to 1000 V, the 1000 V set point voltage is represented by V 1kVRefSetPoint . Similarly, because the actual 1200 V electrode channel set point voltage may not be exactly equal to 1200 V, the 1200 V set point voltage is represented by V 1.2kVSetPoint . In addition, because the voltage of reference channel  202  is lower than the voltage of electrode channels  204 – 210 , no current flows between the reference channel and any of the electrode channels. The output voltage V ChNReadB  at node  222  of each of the electrode channels is measured. 
     The current of each electrode channel is then individually set to a −1.25 μA set point current or I −1 25μASetPoint  while maintaining voltages at node  240  of all other electrode channels at V 1.2kVSetPoint . Because the actual electrode channel set point current may not be exactly equal to −1.25 μA, the −1.25 μA set point current is represented by I −1.25μASetPoint . The electrode channel current setting renders each corresponding diode  226  of the electrode channel forward biased such that voltage at node  240  of the electrode channel is at a voltage equal to the voltage at node  228  of reference channel  202  less a voltage drop across diode  226 . The voltage V ChNReadC  and current I ChNReadC  are measured for each electrode channel. The voltage V RefReadC  and current I RefReadC  are also measured for reference channel  202 . Generally, the reference current is read for each channel reading while the voltage reference is read only once for all the channel readings. 
     Next, the current of each electrode channel is individually set to a −3.75 μA set point current or I −3 75μASetPoint  while maintaining voltages at node  240  of all other electrode channels at V 1 2kVSetPoint . Again, because the actual electrode channel set point current may not be exactly equal to −3.75 μA, the −3.75 μA set point current is represented by I −3 75 μASetPoint . The electrode channel current setting renders each corresponding diode  226  of the electrode channel forward biased such that voltage at node  240  of the electrode channel is at a voltage equal to the voltage at node  228  of reference channel  202  less a voltage drop across diode  226 . The current I ChNReadD  is measured for each electrode channel and the current I RefReadD  from reference channel  202  is also measured. The current of the reference channel is typically measured for each channel current reading. 
     Voltage at node  228  of reference channel  202  is set to a 200 V set point voltage or V 200VRefSetPoint  and voltage at node  228  of all electrode channels are set to a 300 V set point voltage or V 300VSetPoint . Again, because the actual 200 V reference channel set point voltage may not be exactly equal to 200 V, the 200 V set point voltage is represented by V 200VRefSetPoint . Similarly, because the actual 300 V electrode channel set point voltage may not be exactly equal to 300 V, the 300 V set point voltage is represented by V 300VSetPoint . In addition, because the voltage of reference channel  202  is lower than the voltage of the electrode channels, no current flows between the reference channel and any of the electrode channels. The output voltage V ChNReadE  of each of the electrode channels is measured. 
     Lastly, the current of each electrode channel is individually set to a −1.25 μA set point current or I −1.25μSetPoint  while maintaining voltages at node  240  of all other electrode channels at V 300VSetPoint . The electrode channel current setting renders each corresponding diode  226  of the electrode channel forward biased such that voltage at node  228  of the electrode channel is at a voltage equal to the voltage at node  228  of reference channel  202  less a voltage drop across diode  226 . The voltage V ChNReadF  is measured for each electrode channel and the voltage V RefReadF  from reference channel  202  is also measured. Note that the voltage of reference channel is typically measured once for all channel current readings. 
     TABLE I summarizes the calibration steps and the measured voltages and currents of the reference channel and the electrode channels as described above. 
     
       
         
           
               
               
               
             
               
                 TABLE I 
               
             
            
               
                   
               
               
                   
                 Measured Voltage (V) 
                 Measured Current (μA) 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Reference 
                 Electrode 
                 Reference 
                 Electrode 
               
               
                 Calibration Steps 
                 Channel 
                 Channel N 
                 Channel 
                 Channel N 
               
               
                   
               
               
                 (A) Shut off all channels 
                 V RefReadOffset   
                 V ChNReadOffset   
                 I RefReadOffset   
                 I ChNReadOffset   
               
               
                 (B) Set reference channel 
                   
                 V ChNReadB   
               
               
                 to V 1kVRefSetPoint , electrode 
               
               
                 channels to V 1.2kVRefSetPoint   
               
               
                 (C) Set each electrode 
                 V RefReadC   
                 V ChNReadC   
                 I RefReadC   
                 I ChNReadC   
               
               
                 channel to I −1.25μASetPoint   
               
               
                 (D) Set each electrode 
                   
                   
                 I RefReadD   
                 I ChNReadD   
               
               
                 channel to I −3.75μASetPoint   
               
               
                 (E) Set reference channel 
                   
                 V ChNReadE   
               
               
                 to V 200VRefSetPoint , electrode 
               
               
                 channels to V 300VSetPoint   
               
               
                 (F) Set electrode channels 
                 V RefReadF   
                 V ChNReadF   
               
               
                 to I −1.25μASetPoint   
               
               
                   
               
            
           
         
       
     
     The reference channel read gain calibration factors for voltage and current, represented by G RefReadV , G RefReadI , respectively, are known, e.g. calibrated and predetermined at the factory, and utilized in determining various calibration factors and/or offsets. The calibration factors for reading the voltages and currents and the calibration factors and calibration offsets for setting the voltages and currents for each high voltage electrode channel N are shown below as functions of known parameters G RefReadV , G RefReadI  and measured voltages and currents as listed in TABLE I: 
               G   ChNReadV     =       ⁢       G   RefReadV     *       (       V   RefReadC     -     V   RefReadF       )     /                       ⁢     (       V   ChNReadC     -     V   ChNReadF       )                   G   ChNReadI     =       ⁢       G   RefReadI     *       (       I   RefReadD     -     I   RefReadC       )     /                       ⁢     (       I   ChNReadC     -     I   ChNReadD       )                   G   ChNSetV     =       ⁢       G   ChNReadV     *       (       V   ChNReadB     -     V   ChNReadE       )     /                       ⁢     (       V     12   ⁢   k   ⁢           ⁢   VSetPoint       -     V     300   ⁢   VSetPoint         )                   V   ChNSetOffset     =       ⁢       V     300   ⁢   VSetPoint       -       (       V   ChNReadE     -     V   ChNReadOffeset       )     *                       ⁢     (       G   ChNReadV     /     G   ChNSetV       )                   G   ChNSetI     =       ⁢       G   ChNReadI     *       (       I   ChNReadC     -     I   ChNReadD       )     /                       ⁢     (       I       -   125     ⁢           ⁢   μ   ⁢           ⁢   ASetPoint       -     I       -   3.75     ⁢           ⁢   μ   ⁢           ⁢   ASetPoint         )                   I   ChNSetOffset     =       ⁢       I         -   1.25     ⁢           ⁢   μ   ⁢           ⁢   ASetPoint     ⁢               -       (       I   ChNReadC     -     I   ChNReadOffset       )     *                       ⁢     (       G   ChNReadI     /     G   ChNSetI       )               
 
where:
         G ChNReadV  represents the calibration factor for the read voltage gain, i.e. the relation between the reading of voltage  222  and the actual voltage at node  240  for each channel N;   G ChNReadI  represents the calibration factor for the read current gain, i.e. the relation between the reading of current  224  and the actual current at node  240  for each channel N;   G ChNsetV  represents the calibration factor for the voltage setting gain, i.e. the relation between the setting of the DAC set-point output  214  and the actual voltage at node  240  for each channel N;   V ChNSetOffset  represents the offset voltage for setting voltage, i.e., the setting of the DAC set-point output  214  that would result in a 0 voltage at node  240  for each channel N;   G ChNsetI  represents the calibration factor for the current setting gain, i.e. the relation between the setting of the DAC set-point output  214  and the actual current at node  240  for each channel N; and   I ChNSetOffset  represents the offset current for setting current, i.e., the setting of the DAC set-point output  214  that would result in 0 current flow in or out of node  240  for each channel N.       

     In addition, the calibration factor and the voltage offset for setting the voltage for the reference channel are shown below: 
               G   RefSetV     =       ⁢       G   RefReadV     *       (       V   RefReadC     -     V   RefReadF       )     /                       ⁢     (       V     1   ⁢           ⁢   k   ⁢           ⁢   VRefSetPoint       -     V     300   ⁢           ⁢   V   ⁢           ⁢   RefSetPoint         )                   V   RefSetOffset     =       ⁢       V     200   ⁢           ⁢   V   ⁢           ⁢   SetPoint       -       (       V   RefReadF     -     V   RefReadOffeset       )     *                       ⁢     (       G   RefReadV     /     G   RefSetV       )               
 
where:
         G RefSetV  represents the calibration factor for the reference voltage setting gain, i.e., the relation between the setting of the DAC set-point output  214  and the actual voltage at node  228  for the reference channel; and   V RefSetOffset  represents the offset voltage for setting reference voltage, i.e., the setting of the DAC set-point output  214  that would result in a 0 voltage at node  228  for the reference channel.       

     After determining the calibration factors and offsets, the relationships among set point, read back, and output voltages and currents are known. In particular, the actual voltage setting V set, Out  can be expressed as a function of the applied voltage setting V Set  and the actual current setting I Set, Out  can be expressed as a function of the applied current setting I set :
 
Output Voltage= V   ChNOut =( V   set   −V   ChNSetOffset )* G   ChNsetV 
 
Output Current= I   ChNOut =( I   Set   −I   ChNSetOffset )* G   ChNSetI 
 
     In addition, the actual voltage V Read, Out  for each electrode channel can be expressed as a function of the measured voltage V Read  and the actual current I Read, Out  can be expressed as a function of the measured current I Read :
 
Output Voltage= V   ChNOut =( V   Read   −V   ChNReadOffset )* G   ChNReadV 
 
Output Current= I   ChNOut =( I   Read   −I   ChNReadOffset )* G   ChNReadI 
 
     The above-described calibration method is typically generally reduced to generating a first electrical reference input at the reference channel and a first electrical source input at each of the electrode or source channels. A first electrical value at each of the reference and electrode channels are measured. A second electrical reference input at the reference channel and a second electrical electrode input at each of the electrical electrode channels are then generated, the second inputs being different from the corresponding first inputs. A second value at each of the reference and electrical electrode channels are then measured. Each electrical input and each measured value are optionally a voltage and/or a current. 
     A readout calibration factor, e.g., G ChNReadV  or G ChNReadI , is typically determined as a function of a ratio of differences between the first measured reference value and the first measured electrode value and between the second measured reference value and the second measured electrode value. 
     All electrode and reference channels are optionally shut off and an offset voltage and current at each of the reference and electrode channels are measured. A calibration offset value, e.g. V ChNSetOffset  or I ChNSetOffset , is typically determined as a function of the measured offset voltages and currents. In addition, a setting calibration offset V ChNSetOffset  and I ChNSetOffset  are preferably determined as a function of one of the reference inputs and as a function of a difference between one of the measured electrode channel values and one of the measured offset source channel values. 
     An input setting reference calibration offset, e.g., V RefSetOffset , is typically determined as a function of one of the reference inputs and a function of a difference between one of the measured reference channel values and one of the measured offset reference channel values. 
     A setting calibration factor, e.g., G ChNSetV  or G ChNSetI , is typically determined as a function of a ratio of differences between the first measured reference value and the second measured reference value and between the first reference input and the second reference input. 
     A setting reference offset, e.g., G RefsetV , is typically determined as a function a ratio of differences between the first measured reference value and the second measured reference value and between the first reference input and the second reference input. 
     In the calibration process described above, the voltage drop across each of diodes  226  is assumed to be constant at constant current flow such that the diode voltage drops do not have a significant effect on the calibration process because each pair of calibration points is performed at the same bias currents of −1.25 μA. In addition, the offset calibration is not affected by the diode voltage drops because the offset calibration is performed by shutting off all high voltage sources of the reference and electrode channels. 
     Furthermore, the above-described calibration process calibrated a slope of voltage output versus voltage setting assuming a similar voltage drop across each of diodes  226 . The process also ensures against a large voltage difference between the electrode channels during calibration. A large voltage difference between the electrode channels during calibration can generate undesired fluid flow in the microfluidic chip, degrading accuracy and performance. 
     As noted above, any other suitable calibration processes may be utilized and numerous modifications can be optionally made to achieve similar calibration results. For example, the above described calibration process is a two point calibration process such that the process inherently assumes that the circuit components behave linearly, i.e., the circuit components are highly linear and have low voltage coefficients. To compensate for non-linearity circuit components, the above described calibration process may be expanded to perform multiple point calibration for one or more of the calibration factors. 
     While the above is a complete description of preferred embodiments of the invention, various alternatives, modifications, and equivalents can be used. It should be evident that the invention is equally applicable by making appropriate modifications to the embodiments described above. Therefore, the above description should not be taken as limiting the scope of the invention that is defined by the metes and bounds of the appended claims along with their full scope of equivalents.