Patent Publication Number: US-7909424-B2

Title: Method and system for dispensing liquid

Description:
BACKGROUND 
     The abundance of therapeutic targets of drug candidates and of combinatorial and computational technologies has created a demand for laboratory automation of mix-and-measure assays (chemical reaction tests). To increase laboratory productivity and reduce costs, a clear trend towards assay miniaturization, parallelization, and higher throughput has emerged. Traditional approaches to low-volume liquid handling technologies range from classical liquid handlers employing syringe-based dispensing to piezo-electric dispensers. Some offer a fixed volume at the expense of accuracy and precision while others promote a variable volume range at the expense of delivery or dead volume. Most of the pressure syringe-based systems as well as solenoid valve mechanism based systems are not well suited to dispense liquids in nano- to low-micro-liters volume range with great precision, as is required for assay miniaturization demanded by high throughput screening. These traditional dispenser technologies generally comprise an assembly of discrete components, including one nozzle per assembly. Dispensing from a single nozzle can be slow. To compensate partly for the slow throughput performance these single-nozzle dispensers can be multiplexed by adding one or more additional assemblies of discrete components. 
     An inkjet printer typically includes one or more cartridges that contain ink. In some designs, the cartridge has discrete reservoirs of more than one color of ink. Each reservoir is connected via a conduit to a print head that is mounted to the body of the cartridge. The print head is controlled for ejecting minute drops of ink from the print head to a printing medium, such as a paper which is advanced through the printer. The print head is usually scanned across the width of the paper. The paper is advanced, between print head scans, in a direction parallel to the length of the paper. 
     The mechanism for expelling ink drops from each ink chamber (known as a “drop generator”) includes a heat transducer, which typically includes a thin-film resistor. The resistor is carried on an insulated substrate, such as a silicon die. The resistor has conductive traces attached to it so that the resistor can be selectively driven (heated) with pulses of electrical current. The heat from the resistor is sufficient to form a vapor bubble in each ink chamber. The rapid expansion of the bubble propels an ink drop through the nozzle that is adjacent to the ink chamber. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The drawings referenced herein form a part of the specification. Features shown in the drawings are meant as illustrative of exemplary embodiments of the invention. 
         FIG. 1  is a block diagram of an exemplary embodiment of an overall automated liquid handling system incorporating the present invention. 
         FIG. 2  illustrates an automated liquid handling system according to an embodiment of the invention. 
         FIG. 3A  illustrates a perspective view of a modified carriage stand capable of printing on substrates with thickness greater than one (1) centimeter (cm), according to an embodiment of the invention. 
         FIG. 3B  illustrates a plan view of the modified carriage stand of  FIG. 3A , according to an embodiment of the invention. 
         FIG. 4  illustrates an exemplary well-plate which can be filled using the automated liquid handling system of  FIG. 2 . 
         FIG. 5  illustrates a cross-sectional view of a micro machined silicon die which may be used for dispensing liquid into a well-plate of  FIG. 4 . 
         FIG. 6A  illustrates schematically multiple nozzles localized on a single well, according to an embodiment of the present invention. 
         FIG. 6B  illustrates a print head with multiple channels spanning across multiple wells according to an embodiment of the present invention. 
         FIGS. 7A-7D  illustrate a top view and a side view of a well wherein three different liquids have been dispensed and the resultant mixing process, according to an embodiment of the present invention. 
         FIGS. 8A-8C  illustrate an exemplary embodiment of an optical detection system which may be built into the automated liquid handling system of  FIG. 2 . 
         FIG. 9  illustrates an exemplary graphic user interface for dispensing liquid on a well plate, according to an embodiment of the present invention. 
         FIG. 10  illustrates a process flow diagram for selecting printing parameters for the automated liquid handling system of  FIG. 2 , according to an embodiment of the invention. 
         FIGS. 11A-11B  illustrate an exemplary prior art drop detect system which may be incorporated in the system of  FIG. 2 . 
         FIGS. 12A-12G  illustrate another exemplary drop detect system, namely a laser system, which may be incorporated in a system of  FIG. 2 , according to an embodiment of the present invention. 
     
    
    
     DETAILED DESCRIPTION OF THE DRAWINGS 
     In the following detailed description of exemplary embodiments of the invention, reference is made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration exemplary embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized, and logical, mechanical, and other changes may be made without departing from the spirit or scope of the present invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims. 
       FIG. 1  is a block diagram of an overall liquid handling system  100 , according to an embodiment of the present invention. In an exemplary embodiment, liquid handling system  100  is based on an eight (8) color thermal inkjet printer, available as HP Photosmart Pro B9180, from Hewlett Packard, Inc. Liquid handling system  100  can be used for dispensing a liquid on a substrate, for example, on a well plate  102 . Liquid handling system  100  is coupled to a host system  105  (such as a computer or microprocessor) for inputting dispensing parameters such as amount of liquid to be dispensed, number of liquids to be dispensed, and a location on a well plate on which the liquid(s) are to be dispensed. A graphic user interface (see, for example, of  FIG. 9 ) allows a user to dispense desired volumes up to eight (8) different liquids in different mix ratios of volumes ranging from picoliters (pL) to several microliters (μL) for reach liquid. Liquid handling system  100  includes a controller  110 , a power supply  120 , a substrate transport device  125 , a carriage assembly  130  and a plurality of switching devices  135 . The liquid supply device  115  is in fluidic communication with a print head assembly  150  for selectively providing liquids to the print head assembly  150 . The substrate transport device  125  provides a means to move a substrate  102  (such as a well plate) relative to the liquid handling system  100 . Similarly, the carriage assembly  130  supports the print head assembly  150  and provides a means to move the print head assembly  150  to a specific location over the substrate  102  as instructed by the controller  110 . The carriage assembly  130  has been raised to accommodate different well plates or other user specified substrates. The height of carriage assembly  130  with respect to substrate transport device  125  may be adjustable to accommodate substrates of different thicknesses. 
     The print head assembly  150  includes a print head structure  160 . The print head structure  160  contains a plurality of various layers including a substrate ( 510  of  FIG. 5 ). The substrate may be a single monolithic substrate that is made of any suitable material (preferably having a low coefficient of thermal expansion), such as, for example, silicon. The print head structure  160  also includes a high-density arrangement of ink drop generators  165  formed in the print head structure  160  that contains a plurality of elements for causing an ink drop to be ejected from the print head assembly  150 . The print head structure  160  also includes an electrical interface  170  that provides energy to the switching devices  135  that in turn provide power to the high-density arrangement of ink drop generators  165 . 
     During operation of the liquid handling system  100 , the power supply  120  provides a controlled voltage to the controller  110 , the substrate transport device  125 , the carriage assembly  130  and the print head assembly  150 . In addition, the controller  10  receives the dispensing data from the host system  105  and processes the dispensing data into system control information. The dispensing data and other static and dynamically generated data are provided to the substrate transport device  125 , the carriage assembly  130  and the print head assembly  150  for efficiently controlling the liquid handling system  100 . 
       FIG. 2  illustrates an exemplary embodiment of an automated liquid handling system  100 . System  100  includes a substrate transport device  125 , a carriage assembly  130 , a liquid supply device or a pen  115 , and a carrier board  210 . Carrier board  210  carries multiple substrate or well plates  102 . Each pen  115  may accommodate up to two (2) different fluids. Liquids, such as reagents, may be stored in pens  115 , used as needed and then frozen for later use. Pens  115 , therefore, also act as potential storage device for liquids and thus reduce waste of precious liquids which may result from the transfer from a separate storage device. In an exemplary embodiment of the present invention, pens  115  may store the fluids in amounts ranging from ten (10) milliliters (mL) to twenty (20) mL. Smaller stored volumes may be possible, down to less than two (2) mL. Liquid handling system  100  may be encased in an environmental chamber (not shown) to avoid environmental contamination as well as to ensure user safety. An electrostatic drop detect system  1100  is also included to test print head  150  (of  FIG. 1 ) of pens  115 . 
       FIG. 3A  illustrates a perspective view of a carriage stand  300  according to an embodiment of the invention. A substrate transport device  125  carries a carrier board  310 . In an exemplary embodiment, carrier board  310  is constructed with grooves to hold different types of well plates, glass slides and other substrates, for example, nitrocellulose membranes, agar gels, etc. Carrier board  310  carries a well plate  315 . In an exemplary embodiment, carrier board  310  can accommodate up to nine (9) well plates  315 . Carrier board  310  may accommodate substrates having a thickness of one (1) centimeter (cm) or more. A carriage assembly  130  holds inkjet pen  115 . In an exemplary embodiment, carriage assembly  130  holds up to four (4) inkjet pens  115 . Each inkjet pen  115  may contain one (1) or two (2) different fluids. Accordingly, up to eight (8) fluids may be dispensed through four (4) inkjet pens  115  contained by carriage assembly  130 . These exemplary embodiments are non-limiting as different numbers of pens, fluids, plates, and configurations are certainly possible. Carriage assembly  130  travels on a rail  330  which is positioned transversely to carrier board  310 , thus the print heads of pens  115  scan across the width of carrier board  320 . Carrier board  310  is advanced, between print head scans, in a direction parallel to the length of carrier board  310 . Print heads of pens  115  fill well plates  315  in a scanning mode. 
       FIG. 3B  illustrates a plan view of an exemplary embodiment of a carriage stand  300 . Carrier board  310  travels along substrate transport device  125  longitudinally. Carriage assembly  130  travels transversely to carrier board  310 . 
       FIG. 4  illustrates an exemplary well plate  400  suitable for use with system  100 . Well plate  400  has a plurality of wells  410 . An exemplary well plate  400  may have ninety-six (96) wells  410 . Such well plates are known in the art and are available, for example, from Corning Incorporated Life Sciences, Lowell, Mass. Well  410  may accommodate volumes between 190 microliters (μL) to 2000 μL, for example. Other well plates may have different sized wells and different numbers of well compartments. Well  410  may or may not be indented on a substrate and includes any area upon which a liquid is to be dispensed. Common well plates are formatted according to standards, including for example 384 wells, 1536 wells, 2080 wells, 3456 wells, and so on. Plates having more wells also typically have a smaller well size and well volume, down to approximately 1 uL and smaller. It is to be understood that embodiments of the present invention are compatible with the smallest wells by dispensing in the nano- to low-micro-liter volume range. 
       FIG. 5  illustrates a micro machined silicon die  500  through which liquid is dispensed into, for example, a well  410  ( FIG. 4 ). A slot  525  is machined in a silicon substrate  510 . In another embodiment of the present invention, substrate  510  may be glass or other insulating material preferably with a low coefficient of thermal expansion. By way of non-limiting example only, the slot may have a narrow opening ranging from about 0.05 millimeter (mm) to 0.5 mm. In an exemplary embodiment, substrate  510  may have a thickness ranging from about 300 micrometers (μm) to 2000 μm. A polymer layer  515  is deposited over substrate  510 . Polymer layer  515  may have thickness ranging from about 10 μm to about 60 μm. In an exemplary embodiment, two apertures  520  are formed in polymer layer  515 . Apertures  520  act as nozzles through which liquid is dispensed. In an exemplary embodiment of the present invention, apertures  520  have diameters ranging from about 5 μm to about 100 μm. Two resistors  521  are provided for two apertures  520 . Resistors  521  provide heat to form bubbles of the liquids and to expel the liquid bubbles out of apertures  520 . In an exemplary embodiment of the invention, a substrate  510  may have one hundred (100) to two thousand (2000) apertures. Liquid to be dispensed travels through slot  525  and is dispensed through apertures  520  as is known in the art. Drops of liquids in size ranging from about 5 picoliter (pL) to 200 pL may be fired from a nozzle  520  at a frequency ranging from 1 kilohertz (kHz) to 20 kHz. Since the actuation mechanism is built in close proximity to nozzles  520 , there is no requirement for a large fluid head for reproducible ejection, and the liquid waste is reduced down to nanoliters (nL). 
       FIG. 6A  illustrates a schematic side view of a well  410 . A typical thermal inkjet print head (not shown) is designed at 1200 dots per inch (dpi) in the paper axis. A typical well  410  may have an opening of about 2 millimeter (mm). Thus, up to 100 nozzles may be localized on well  410  at any given instant. The swath height of the typical inkjet print head  160  (of  FIG. 6B ) is about one (1) inch. Thus, the entire swath of the print head covers about six (6) wells of about 2 mm diameter at any given instant. Accordingly, a plurality of nozzles may be localized on a single well  410 , as well as a plurality of nozzles may be localized on a plurality of wells  410 . Controller  110  (of  FIG. 1 ) contains computer code which uses a digital “half-toning” writing systems routine to create dispersion of drops in individual wells  410 . Three nozzles  520   a ,  520   b , and  520   c  are localized on well  410 . Drops  605   a ,  605   b , and  605   c  are dispensed by nozzles  520   a ,  520   b , and  520   c  respectively into well  410 . Drops  605   a ,  605   b , and  605   c  may have diameters ranging from about 10 μm to 100 μm. The volume of liquid dispensed in a single drop is typically a fraction of the total desired volume. Hence, the dispensing step is repeated multiple times until the desired volume of a given liquid is dispensed. For each type of liquid, a look up table contains the drop volume and firing parameters. Each layer of drops will have approximately the same thickness as that of the drop diameter. The diffusion distance for individual molecules of drops  520   a ,  520   b , and  520   c  is relatively small when compared to a drop having a diameter of 1 mm such as may be dispensed by conventional single-nozzle technologies. Here, instead, the mixing of individual molecules from within drops  520   a ,  520   b , and  520   c  is greatly enhanced by the drops having small size, being dispensed with some finite velocity, and being dispensed as layers. If more than one liquid is to be dispensed, each liquid may be alternately dispensed in well  410 , which facilitates rapid mixing of different liquids. Multiple layers of multiple liquids may therefore be alternately dispensed in well  410 . 
       FIG. 6B  illustrates four print heads  160  spanning over multiple rows and multiple columns of wells  410 . Each print head  160  has two channels  610  and  620 , each of which can dispense a distinct liquid. 
     Referring now to  FIG. 7A , a top view of well  410  is illustrated. Dispensing and mixing of three different fluids is described only by way of a non-limiting example. In a first stage of dispensing, two different fluids  705  and  710  are dispensed sequentially in well  410 . Nozzles  520   a  and  520   b  (of  FIG. 6 ) may be positioned such that each following nozzle is slightly offset from the firing position of the previous nozzle.  FIG. 7B  illustrates a side view of well  410 . Two liquids  705  and  710  have been dispensed sequentially in well  410 . Since drop sizes may be as small as 10 μm to 100 μm, mix time is relatively short and the mixing of different liquids is almost instantaneous. Liquids  705  and  710  mix into a generally homogeneous mixture  720 . Then a third liquid  715  is dispensed in well  410 , as shown in  FIG. 7C . Third liquid  715  mixes with mixture  720  and forms a generally homogeneous mixture  730 , as shown in  FIG. 7D . As will be understood by one skilled in the art, more than three liquids may be dispensed in well  410 , and different dispensed liquids will form a generally homogeneous mixture in well  410  in a similar fashion. The mixing time of different liquids will depend on a multitude of factors such as the molecular structures of different liquids, the temperature of the liquids, presence of any solvents or co-solvents, ionic strengths of different liquids and the pH factors of different liquids. The mixing may be greatly enhanced by the small drop size and the layered dispensing. By way of a non-limiting example, a typical color dye in a low surface tension fluid, such as a typical inkjet ink, the mixing is almost instantaneous. However, for liquids with larger molecules, the diffusion time may be relatively longer. Even then, the mixing is comparatively faster than in case of the methods currently employed to dispense such liquids. 
       FIG. 8A  illustrates an exemplary embodiment of an optical sensor  800 . Optical sensor  800  includes a light source  810  and a Light to Voltage (LTV) converter  820 . Light source  810  may include a multiple number of Light Emitting Diodes (LED) of different colors, in an exemplary embodiment of the present invention. As illustrated in  FIG. 8B , optical sensor  800  is positioned on carriage assembly  130 . Optical sensor  800  may be in close proximity of pens  115  (of  FIG. 2 ).  FIG. 8C  illustrates schematically the operating principle of optical sensor  800 . Light emitted by light source(s)  810  is focused on a well  410 , wherein one or more liquids have been dispensed. The light reflected by the one or more liquids in well  410  is sensed by converter  820 . Voltage generated by converter  820  is a function of the type of light and the type and volume of liquid(s) present in well  410 . Since the type of light is known, the type and volume of liquid(s) dispensed in well  410  may be determined. Optical sensor  800  may be used after every pass of carriage assembly  130  (of  FIG. 2 ) over well  410 , or after liquid has been dispensed in the entire well plate  315  (of  FIG. 3A ), for example. Optical sensor  800  may also be used to measure absorbance, fluorescence and luminescence of the mixture of liquid dispensed in well  410 . 
       FIG. 9  illustrates an exemplary graphic user interface  900 . Interface  900  displays the layout  905  of a well plate  400 . Each well  950  of well plate  400  is graphically represented in interface  900 . In an exemplary embodiment of the present invention, well  950  may be selected by clicking on layout  905  using an input device such as a mouse. Each well  950  is uniquely identified and the selected well  950  is displayed in a text box  945 . Alternatively, well  950  may also be selected by typing in a unique identifier in another embodiment. Each of up to eight (8) different liquids may be graphically represented in a distinct color in a legend box  940 . A liquid may be selected to be dispensed in a desired well  950  using a pull-down menu  910 . The desired volume of the selected liquid to be dispensed may be input in a text box  915 . Once the liquid and the desired volume are selected, button  920  may be clicked to add the selection to the list displayed in a text box  955 . Each selected liquid for a given well  950  is graphically represented in display box  930  which represents the selected well  950 . Text box  955  displays each of the selected liquids and their respective volumes which are to be dispensed in selected well  950 . Buttons  960  may be used to select and remove a previously selected liquid for given well  950 , if it is no longer desired to dispense that liquid. A given selection of liquids and their volumes to be dispensed may be saved for future use using the buttons in a box  935 . Multiple numbers of well plates may be graphically represented using radio buttons  965 . Clicking on button  925  will cause the liquid(s) to be dispensed onto the well plate(s) previously selected. 
     Referring now to  FIG. 10 , an exemplary process flow is illustrated. At block  1005   a , the user selects a well plate from a library of well plate types. Responsive to the user selection, the computer code identifies margins and well positions from a look-up table, based on the selected well plate type, as at block  1005   b . The user identifies a channel and selects a fluid type for the channel from the library of fluid types, as at block  1010   a . At block  1010   b , responsive to the fluid selection by the user, the computer code identifies drop volume and firing parameters from a look-up table based on the selected fluid type. The computer code causes a graphical representation of the selected well plate with selectable wells to be shown to the user, as at block  1015   b . At block  1015   a , the user selects the wells in which it is desired to add fluid, using the graphical interface, in an exemplary embodiment of the invention. The user inputs the volume of fluid to be dispensed to the selected wells, at block  1020   a . Responsive to the user input, the computer code calculates the number of drops of fluids, number of passes required and the drop positions within a well to dispense the volume of fluid as desired by the user, at block  1020   b . At block  1025 , the system confirms if all desired fluids have been selected for the available channels. If all the desired fluids have not been selected, the steps depicted in blocks  1010   a - 1010   b  to blocks  1020   a - 1020   b  are repeated until all the desired fluids have been selected. At block  1030 , the user issues a command to start dispensing the fluid(s) as per the selected parameters. 
       FIG. 11A  illustrates an exploded perspective view of an exemplary electrostatic drop detect system  1100  for system  100  of  FIG. 1 . Drop detect system  1100  includes a bias plate  1120 , a sensing plate  1110 , and a holder  1130 . Holder  1130  collects fluid drops dispensed into drop detect system  100 . Referring now to  FIG. 11B , a print head  160  is positioned above drop detect system  1100 . A fluid drop  1140  is fired from print head  160 . Fluid drop is electrostatically charged by bias plate  1120 . Sensing plate  1110  detects the voltage of the charged fluid drop  1140 . Based on the measured voltage of fluid drop  1140 , the volume of fluid drop  1140  may be determined. Drop detect system  1100  may be used to test a nozzle  500  (of  FIG. 5 ) of print head  160  and check whether a proper volume of fluid is fired from nozzle  500 . If no fluid drop  1140  is detected, or if volume of fluid drop  1140  varies from the desired volume, nozzle  500  may be clogged or malfunctioning. In such a case, other functioning nozzles may be used to dispense the desired volume of fluid. Since system  100  (of  FIG. 1 ) may have hundreds of nozzles  500  for a single fluid, the impact of any clogged or malfunctioning nozzles would be minimal, as other properly functioning nozzles may be used to dispense the required amount of liquid. In an exemplary embodiment of the present invention, all print heads  160  may be tested using drop detect system  1100  either before the dispensing of liquids is undertaken or periodically in between passes over substrate  102  (of  FIG. 1 ). 
       FIGS. 12A-12F  illustrate an exemplary embodiment of a laser system  1200 , which may be incorporated in liquid handling system  100  of  FIG. 2 . Laser system  1200  includes two laser emitters  1210  and  1230  and two laser detectors  1220  and  1240 . Laser beam emitted by emitter  1210  is detected by detector  1220  and laser beam emitted by emitter  1230  is detected by detector  1240 . The first pair of emitter  1210  and detector  1220  is separated from the second pair of emitter  1230  and detector  1240  by a predetermined distance x.  FIG. 12A  shows a drop  1140  (of  FIG. 11 ) fired by a print head  160  (of  FIG. 1 ). Drop  1140  has not yet intercepted either of the laser beams emitted by emitters  1210  and  1230 . In  FIG. 12B , drop  1140  has just intercepted the laser beam emitted by emitter  1210 . The time t 1a  when drop  1140  has just intercepted the laser beam is recorded. In  FIG. 12C , drop  1140  is about to leave the pathway of laser beam emitted by emitter  1210 . The time t 1b  is recorded when detector  1220  detects the laser beam emitted by emitter  1210 . Referring now to  FIG. 12D , drop  1140  has just intercepted the laser beam emitted by emitter  1230 . The time t 2a  is recorded when drop  1140  has just intercepted the laser beam emitted by emitter  1230 . In  FIG. 12E , drop  1140  is about to leave the pathway of laser beam emitted by emitter  1230 . Time t 2b  is recorded when detector  1240  detects the laser beam emitted by emitter  1230 . In FIG.  12 F, drop  1140  is collected in a gutter  1250 , and both detectors  1220  and  1240  detect the laser beams emitted by emitters  1210  and  1230  respectively. 
     Since the predetermined distance x between the two pairs of emitter-detectors is known and the time taken by drop  1140  to travel the distance between the two pairs of emitter-detector is known, the velocity of drop  1140  can be calculated as follows:
 
Drop Velocity= x /( t   2a   −t   1a )
 
As shown in  FIG. 12G , the time interval between t 1a  and t 1b  may be used to deduce the drop volume. Time interval (t 1a −t 1b ) indicates the time taken by drop  1140  to pass through a distance approximately equal to the diameter of drop  1140 . Since the time interval (t 1a −t 1b ) is known and the drop velocity can be calculated as above, the distance can be calculated. The volume of drop  1140  may be determined from the diameter of drop  1140 , and if the density of the liquid is known, drop weight of drop  1140  may also be determined. Correspondingly smaller volumes of the two illustrated satellite drops may also be calculated.
 
     An exemplary application of liquid handling system  100  is to precisely dispense liquids for chemical reaction tests, for example, in preparing mix-and-measure assays. By lowering the total volume of chemical reagents and living cells used in such assays, the cost may be decreased. Systems are known in the art to dispense liquids in volumes as large as 300 μL to as small as 2-10 μL. Since liquid handling system  100  is capable of dispensing liquid in form of drops as small as 5 pL to 200 pL, the assay volumes may be reduced from microliters to nanoliters without compromising on precision. Although dispense times are highly application-dependent, in an exemplary embodiment, a combination of up to eight (8) liquids may be dispensed in six (6) well plates having 1536 wells in approximately one (1) minute. Since pen  115  (of  FIG. 1 ) may store up to twenty (20) mL of a fluid, numerous assays may be prepared before pen  115  needs to be replaced or refilled, thus cutting down assay preparation time. 
     It is noted that, although specific embodiments have been illustrated and described herein, it will be appreciated by those of ordinary skill in the art that any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This application is thus intended to cover adaptations or variations of the disclosed embodiments of the present invention. Therefore, it is intended that this invention be limited only by the claims and equivalents thereof.