Patent Publication Number: US-6220075-B1

Title: Method for determining and verifying a microvolume of a sample liquid dispersed in droplets

Description:
This application is a divisional of Ser. No. 09/012,174 filed Jan. 22, 1998 which is a continuation of Ser. No. 08/656,455 filed May 31, 1996 and now abandoned. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to an apparatus and process for controlling, dispensing and measuring small quantities of fluid. More specifically, the present invention senses pressure changes to ascertain and confirm fluid volume dispensed and proper system functioning. 
     BACKGROUND OF THE INVENTION 
     Advances in industries employing chemical and biological processes have created a need for the ability to accurately and automatically dispense small quantities of fluids containing chemically or biologically active substances for commercial or experimental use. Accuracy and precision in the amount of fluid dispensed is important both from the standpoint of causing a desired reaction and minimizing the amount of materials used. 
     Equipment for dispensing microvolumes of liquid have been demonstrated with technologies such as those developed for ink jet applications. However, ink jet equipment has the advantage of operating with a particular ink (or set of inks) of known and essentially fixed viscosity and other physical properties. Thus, because the properties of the ink being used are known and fixed, automatic ink jet equipment can be designed for the particular ink specified. Direct use of ink jet technology with fluids containing a particular chemical and biological substance of interest (“transfer liquid”) is more problematic. Such transfer liquids have varying viscosity and other physical properties that make accurate microvolume dispensing difficult. Automatic microvolume liquid handling systems should be capable of handling fluids of varying viscosity and other properties to accommodate the wide range of substances they must dispense. Another aspect of this problem is the need to accommodate accurately dispensing smaller and smaller amounts of transfer liquid. Especially in the utilization and test of biological materials, it is desirable to reduce the amount of transfer liquid dispensed in order to save costs or more efficiently use a small amount of material available. It is often both desirable and difficult to accurately dispense microvolumes of transfer liquid containing biological materials. Knowing the amount of transfer liquid dispensed in every ejection of transfer liquid would be advantageous to an automated system. 
     Another difficulty with dispensing microvolumes of transfer liquid arises due to the small orifices, e.g., 20-80 micrometers in diameter, employed to expel a transfer liquid. These small orifice sizes are susceptible to clogging. Further exacerbating the clogging problem are the properties of the substances sometimes used in the transfer liquid. Clogging of transfer liquid substances at the orifice they are expelled from, or in other parts of the dispenser, can halt dispensing operations or make them far less precise. Therefore, it would be desirable to be able to detect when such conditions are occurring, and to be able to automatically recover from these conditions. Failure of a microvolume dispenser to properly dispense transfer fluid can also be caused by other factors, such as air or other compressible gases being in the dispensing unit. It would be desirable to detect and indicate when a microvolume dispenser is either not dispensing at all, or not dispensing the desired microvolume (“misfiring”). 
     In order to achieve an automated microvolume dispensing system it would be desirable to ensure in realtime that the transfer liquid is within some given range of relevant system parameters in order to rapidly and accurately dispense transfer liquid droplets of substantially uniform size. Because industry requires rapid dispensing of microvolume amounts of transfer liquid, it is desirable to be able to ascertain transfer liquid volume dispensed, and to be able to detect and recover from dispensing problems in realtime. 
     SUMMARY OF THE INVENTION 
     It is a primary object of the present invention to provide a microvolume liquid handling system which is capable of accurately verifying microvolume amounts of transfer liquid dispensed by sensing a corresponding change in pressure in the microvolume liquid handling system. 
     It is also an object of the present invention to provide a microvolume liquid handling system which can accurately measure an amount of dispensed liquid regardless of transfer liquid properties such as viscosity. 
     It is another object of the present invention to provide a microvolume liquid handling system which can transfer microvolume quantities of fluids containing chemically or biologically active substances. 
     It is still another object of the present invention to provide a microvolume liquid handling system which senses pressure changes associated with clogging and misfiring to indicate such improper operation. 
     It is yet another object of the present invention to provide a microvolume liquid handling system which can verify that the transfer liquid is maintained within a given range of negative pressure (with respect to ambient atmospheric pressure) in order to accurately dispense microvolume amounts of transfer liquid and optimize the operation of the microdispenser. 
     Other objects and advantages of the present invention will be apparent from the following detailed description. 
     Accordingly, the foregoing objectives are realized by providing a microvolume liquid handling system which includes a positive displacement pump operated by a stepper motor, a piezoresistive pressure sensor, and an electrically controlled microdispenser that utilizes a piezoelectric transducer bonded to a glass capillary. The microdispenser is capable of rapidly and accurately dispensing sub-nanoliter (“nl”) sized droplets by forcibly ejecting the droplets from a small nozzle. 
     To provide the functionality of an automated liquid handling system, the microdispenser is mounted onto a 3-axis robotic system that is used to position the microdispenser at specific locations required to execute the desired liquid transfer protocol. 
     The present invention includes a system liquid and a transfer liquid in the dispensing system separated by a known volume of air (“air gap”) which facilitates measuring small changes in pressure in the system liquid that correlate to the volume of transfer liquid dispensed. The transfer liquid contains the substances being dispensed, while in one preferred embodiment the system liquid is deionized water. Each time a droplet in the microvolume dispensing range is dispensed, the transfer liquid will return to its prior position inside the microdispenser because of capillary forces, and the air gap&#39;s specific volume will be increased corresponding to the amount of transfer liquid dispensed. This has the effect of decreasing pressure in the system liquid line which is measured with a highly sensitive piezoresistive pressure sensor. The pressure sensor transmits an electric signal to control circuitry which converts the electric signal into a digital form and generates an indication of the corresponding volume of transfer liquid dispensed. An advantage of the present invention is its insensitivity to the viscosity of the transfer liquid. This is because the pressure change in the system liquid corresponds to the microvolume dispensed, without being dependent on the dispensed fluid viscosity. The present invention possesses unique capabilities in microvolume liquid handling. This system is capable of automatically sensing liquid surfaces, aspirating liquid to be transferred, and then dispensing small quantities of liquid with high accuracy, speed and precision. The dispensing is accomplished without the dispenser contacting the destination vessel or contents. A feature of the present invention is the capability to positively verify the microvolume of liquid that has been dispensed during realtime operation. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a block diagram of the a microvolume liquid handling system embodying the present invention; 
     FIG. 2 is a schematic of a positive displacement pump; 
     FIG. 3 is an illustration of a microdispenser and a piezoelectric transducer; and 
     FIG. 4 is a graph depicting the system line pressure during a microdispenser dispense. 
    
    
     While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that it is not intended to limit the invention to the particular forms disclosed. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Turning now to the drawings and referring first to FIG. 1, a microvolume liquid handling system  10  is illustrated. The microvolume liquid handling system  10  includes a positive displacement pump  12 , a pressure sensor  14  and a microdispenser  16 . Tubing  18  connects the positive displacement pump  12  to the pressure sensor  14  and the pressure sensor  14  to the microdispenser  16 . The positive displacement pump  12  moves a system liquid  20  through the pressure sensor  14  and the microdispenser  16 . After the system  10  is loaded with system liquid  20 , an air gap  22  of known volume, then an amount of transfer liquid  24 , are drawn into the microdispenser  16  in a manner described below. The transfer liquid  24  contains one or more biologically or chemically active substances of interest In one preferred embodiment the microdispenser  16  expels (or synonymously, “shoots”) sub-nanoliter size individual droplets  26  which are very reproducible. The expelled droplets  26  of transfer liquid  24  are on the order of 0.45 nanoliters per droplet  26  in one preferred embodiment, but they can be as small as 5 picoliters. For example, if one desires to expel a total of 9 nanoliters of transfer liquid  24 , then the microdispenser  16  will be directed to expel 20 droplets  26 . Droplet  26  size can be varied by varying the magnitude and duration of the electrical signal applied to the microdispenser  16 . Other factors affecting droplet size include: the size of the nozzle opening at the bottom of the microdispenser, the pressure at the microdispenser inlet, and properties of the transfer liquid. 
     Referring now to FIGS. 1 and 2, in one preferred embodiment the positive displacement pump  12  is a XL 3000 Modular Digital Pump manufactured by Cavro Scientific Instruments, Inc., 242 Humboldt Court, Sunnyvale, Calif. 94089. The positive displacement pump  12  includes stepper motor  28  and stepper motor  29 , and a syringe  30 . The syringe  30  includes a borosilicate glass tube  32  and a plunger  34  which is mechanically coupled through a series of gears and a belt (not shown) to the stepper motor  28 . Stepper motor  28  motion causes the plunger  34  to move up or down by a specified number of discrete steps inside the glass tube  32 . The plunger  34  forms a fluidtight seal with the glass tube  32 . In one preferred embodiment syringe  30  has a usable capacity of 250 microliters which is the amount of system liquid  20  the plunger  34  can displace in one fill stroke. Depending on the selected mode of operation, the stepper motor  28  is capable of making 3,000 or 12,000 discrete steps per plunger  34  full stroke. In one preferred embodiment the stepper motor  28  is directed to make 12,000 steps per full plunger  34  stroke with each step displacing approximately 20.83 nanoliters of system liquid  20 . In one preferred embodiment the system liquid  20  utilized is deionized water. 
     Digitally encoded commands cause the stepper motor  28  within the positive displacement pump  12  to aspirate discrete volumes of liquid into the microdispenser  16 , wash the microdispenser  16  between liquid transfers, and to control the pressure in the system liquid  20  line for microvolume liquid handling system  10  operation. The positive displacement pump  12  is also used to prime the system  10  with system liquid  20  and to dispense higher volumes of liquid through the microdispenser  16 , allowing dilute solutions to be made. The positive displacement pump  12  can also work directly with transfer liquid  24 . Thus, if desired, transfer liquid  24  can be used as system liquid  20  throughout the microvolume liquid handling system  10 . 
     To prime the microvolume liquid handling system  10 , the control logic  42  first directs a 3-axis robotic system  58  through electrical wire  56  to position the microdispenser  16  over a wash station contained on the robotic system  58 . In one preferred embodiment the microvolume liquid handling system  10  includes, and is mounted on, a 3-axis robotic system is a MultiPROBE CR10100, manufactured by Packard Instrument Company, Downers Grove, Ill. The positive displacement pump  12  includes a valve  38  for connecting a system liquid reservoir  40  to the syringe  30 . An initialization control signal is transmitted through the electrical cable  36  to the pump  12  by control logic  42  which causes the valve  38  to rotate connecting the syringe  30  with the system fluid reservoir  40 . The control signal also causes the stepper motor  28  to move the plunger  34  to its maximum extent up (Position  1  in FIG. 2) into the borosilicate glass tube  32 . The next command from the control logic  42  causes the stepper motor  28  to move the plunger  34  to its maximum extent down (Position  2  in FIG. 2) inside the tube  32 , to extract system liquid  20  from the system reservoir  40 . Another command from the control logic  42  directs the valve  38  to rotate again, causing the syringe  30  to be connected with the tubing  18  connected to the pressure sensor  14 . In one referred embodiment the tubing  18  employed in the microvolume liquid handling system  10  is Natural Color Teflon Tubing made by Zeus Industrial Products, Inc., Raritan, N.J., with an inner diameter of 0.059 inches and an outer diameter of 0.098 inches. The next command from the control logic  42  to the positive displacement pump  12  causes the system liquid  20  inside of the syringe  30  to be pushed into the microvolume liquid handling system  10  towards the pressure sensor  14 . Because the microvolume liquid handling system  10  typically requires about 4 milliliters of system fluid to be primed, the sequence of steps described above must be repeated about 16 times in order to completely prime the microvolume liquid handling system  10 . 
     The control logic  42  receives signals from the pressure sensor  14  through an electrical line  46 . The signals are converted from an analog form into a digital form by an A/D (analog to digital) converter  44  and used by the control logic  42  for processing and analysis. In one preferred embodiment the A/D conversion is a PC-LPM-16 Multifunction I/O Board manufactured by National Instruments Corporation, Austin, Tex. At various points in the liquid transfer process described herein, the control logic  42  receives signals from the pressure transducer  14 , and sends command signals to the pump  12 , microdispenser electronics  51 , and the 3-axis robotic system  58 . Within the control logic  42  are the encoded algorithms that sequence the hardware (robotic system  58 , pump  12 ,and microdispenser electronics  51 ) for specified liquid transfer protocols as described herein. Also within the control logic  42  are the encoded algorithms that process the measured pressure signals to: verify and quantify microdispenses, perform diagnostics on the state of the microvolume liquid handling system, and automatically perform a calibration of the microdispenser for any selected transfer liquid  24 . 
     The pressure sensor  14  senses fluctuations in pressure associated with priming the microvolume liquid handling system  10 , aspirating transfer liquid  24  with pump  12 , dispensing droplets  26  with microdispenser  16 , and washing of microdispenser  16  using pump  12 . In one preferred embodiment the pressure sensor  14  is a piezoresistive pressure sensor part number 26PCDFG6G, from Microswitch, Inc., a Division of Honeywell, Inc., 11 West Spring Street, Freeport, Ill. 61032. Also included with the pressure sensor  14  in the block diagram in FIG. 1 is electrical circuitry to amplify the analog pressure signal from the pressure sensor. The pressure sensor  14  converts pressure into electrical signals which are driven to the A/D converter  44  and then used by the control logic  42 . For example, when the microvolume liquid handling system  10  is being primed, the pressure sensor  14  will send electrical signals which will be analyzed by the control logic  42  to determine whether they indicate any problems within the system such as partial or complete blockage in the microdispenser  16 . 
     Once the microvolume liquid handling system  10  is primed, the control logic  42  sends a signal through electrical wire  56  which instructs the robotic system  58  to position the microdispenser  16  in air over the transfer liquid  24 . The control logic  42  instructs stepper motor  28  to move the plunger  34  down, aspirating a discrete quantity of air (air gap), e.g., 50 microliters in volume into the microdispenser  16 . The control logic  42  then instructs the robotic system  58  to move the microdispenser  16  down until it makes contact with the surface of the transfer liquid  24  (not shown) is made. Contact of the microdispenser  16  with the surface of the transfer liquid  24  is determined by a capacitive liquid level sense system (U.S. Pat. No. 5,365,783). The microdispenser is connected by electrical wire  55  to the liquid level sense electronics  54 . When the liquid level sense electronics  54  detects microdispenser  16  contact with transfer liquid  24  surface, a signal is sent to the robotic system  58  through electrical wire  53  to stop downward motion. 
     The control logic  42  next instructs the pump  12  to move the plunger  34  down in order to aspirate transfer liquid  24  into the microdispenser  16 . The pressure signal is monitored by control logic  42  during the aspiration to ensure that the transfer liquid  24  is being successfully drawn into the microdispenser  16 . If a problem is detected, such as an abnormal drop in pressure due to partial or total blockage of the microdispenser, the control logic  24  will send a stop movement command to the pump  12 . The control logic  24  will then proceed with an encoded recovery algorithm. Note that transfer liquid  24  can be drawn into the microvolume liquid handling system  10  up to the pressure sensor  14  without threat of contaminating the pressure sensor  14 . Additional tubing can be added to increase transfer liquid  24  capacity. Once the transfer liquid  24  has been aspirated into the microdispenser  16 , the control logic  42  instructs the robotic system  58  to reposition the microdispenser  16  above the chosen target, e.g., a microtitre plate. 
     In one preferred embodiment the microdispenser  16  is the MD-K-130 Microdispenser Head manufactured by Microdrop, GmbH, Muhlenweg 143, D-22844 Norderstedt, Germany. 
     As illustrated in FIG. 3, the microdispenser  16  consists of a piezoceramic tube  60  bonded to a glass capillary  62 . The piezoceramic tube has an inner electrode  66  and an outer electrode  68  for receiving analog voltage pulses which cause the piezoceramic tube to constrict. Once the glass capillary  62  has been filled with transfer liquid  24 , the control logic  42  directs the microdispenser electronics  51  by electrical wire  50  to send analog voltage pulses to the piezoelectric transducer  60  by electrical wire  52 . In one preferred embodiment the microdispenser electronics  51  is the MD-E-201 Drive Electronics manufactured by Microdrop, GmbH, Muhlenweg 143, D-22844 Norderteedt, Germany. The microdispenser electronics  51  control the magnitude and duration of the analog voltage pulses, and also the frequency at which the pulses are sent to the microdispenser  16 . Each voltage pulse causes a constriction of the piezoelectric transducer  60 , which in turn deforms the glass capillary  62 . The deformation of the glass capillary  62  produces a pressure wave that propagates through the transfer liquid  24  to the microdispenser nozzle  63  where one droplet  26  of transfer liquid  24  is emitted under very high acceleration. The size of these droplets  26  has been shown to be very reproducible. The high acceleration of the transfer liquid  24  minimizes or elinnnates problems caused by transfer liquid  24  surface tension and viscosity, allowing extremely small droplets  26  to be expelled from the nozzle, e.g., as small as 5 picoliter droplets  26  have been demonstrated. Use of the microdispenser  16  to propel droplets  26  out of the nozzle also avoids problems encountered in a liquid transfer technique called touchoff. In the touchoff technique, a droplet  26  is held at the end of the nozzle and is deposited onto a target surface by bringing that droplet  26  into contact with the target surface while it is still hanging off of the microdispenser  16 . Such a contact process is made difficult by the surface tension, viscosity and wetting properties of the microdispenser  16  and the target surface which lead to unacceptable volume deviations. The present invention avoids the problems of the contact process because the droplets  26  are expelled out of the microdispenser  16  at a velocity of several meters per second. The total desired volume is dispensed by the present invention by speciying the number of droplets  26  to be expelled. Because thousands of droplets  26  can be emitted per second from the microdispenser  16 , the desired microvolume of transfer liquid  24  can rapidly be dispensed. 
     In one preferred embodiment, the lower section of the glass capillary  62 , between the piezoelectric transducer  60  and the nozzle  63 , is plated with a conductive material, either platinum or gold. This provides an electrically conductive path between the microdispenser  16  and the liquid level sense electronics  54 . In one preferred embodiment the glass capillary  62  has an overall length of 73 millimeters, and the nozzle  63  has an internal diameter of 75 micrometers. 
     To dispense microvolume quantities of transfer liquid  24 , analog voltage pulses are sent to the microdispenser  16 , emitting droplets  26  of liquid. Capillary forces acting on the transfer liquid  24  replace the volume of transfer liquid  24  emitted from the microdispenser  16  with liquid from the tubing  18 . However, since the transfer liquid-air gap-system liquid column terminates at a closed end in the positive displacement pump  12 , there is a corresponding drop in the system liquid  20  line pressure as the air gap  22  is expanded. This is illustrated in FIG. 4 which depicts the pressure profile measured during a microdispense of 500 nanoliters. Important to the present invention, the magnitude of the pressure drop is a function of the size of the air gap  22  and the volume of the liquid dispensed. 
     With an air gap  22  of known volume, the pressure change as detected by the pressure sensor  14  relates to the volume dispensed. Thus, the control logic  42  determines from the pressure change measured by the pressure sensor  14 , the volume of transfer liquid  24  that was dispensed. In one preferred embodiment of the present invention it is preferable that the drop in pressure not exceed approximately 30 to 40 millibars below ambient pressure, depending on the properties of the transfer liquid  24 . If the amount of transfer liquid  24  dispensed is sufficient to drop the pressure more than 30 to 40 millibars, the pressure difference across the microdispenser  16 , i.e., between the ambient pressure acting on the nozzle  63  and the pressure at the capillary inlet  63 , will be sufficient to force the transfer liquid  24  up into the tubing  18 . This will preclude firer dispensing. There is a maximum amount of transfer liquid  24  that can be dispensed before the control logic  42  is required to command the pump  12  to advance the plunger  34  to compensate for the pressure drop. This maximum volume is determined by the desired dispense volume and the size of the air gap  22 . Conversely, the size of the air gap  22  can be selected based on the desired dispense volume so as not to produce a pressure drop exceeding 30 to 40 millibars below ambient pressure. It is also within the scope of the present invention to advance the plunger  34  while the microdispenser  16  is dispensing, thereby rebuilding system liquid  20  line pressure, so that the microdispenser  16  can operate continuously. 
     The change in system liquid  20  pressure is used to determine that the desired amount of transfer liquid  24  was dispensed. A second verification of the amount of A. transfer liquid  24  that was dispensed is made by the control logic  42  monitoring the system liquid  20  line pressure while directing the pump  12  to advance the syringe plunger  34  upwards towards Position  1 . The syringe plunger  34  is advanced until the system liquid  20  line pressure returns to the initial (pre-dispense) value. By the control logic  42  tracking the displaced volume the plunger  34  moves (20.83 nanoliters per stepper motor  28  step), a second confirmation of dispensed volume is made, adding robustness to the system. The system liquid  20  line pressure is now at the correct value for the next microdispenser  16  dispense, if a multi-dispense sequence has been specified. 
     Once the transfer liquid  24  dispensing has been completed, the control logic  24  causes the robotic system  58  to position the microdispenser  16  over the wash station. The control logic  24  then directs pump  12  and robotic system  58  in a wash sequence that disposes of any transfer liquid  24  left in the microdispenser  16 , and washes the internal surface of glass capillary  62  and the external surface in the nozzle  63  area that was exposed to transfer liquid  24 . The wash fluid can either be system liquid  20  or any other liquid placed onto the deck of the robotic system  58 . The wash sequence is designed to minimize cross-contamination of subsequent transfer liquids  24  with transfer liquids processed prior. Toward this end, it is also possible to enable an ultrasonic wash of the microdispenser  16 . This is accomplished by the control logic  42  directing the microdispenser electronics  51  to send electrical pulses to the microdispenser at a frequency in the ultrasonic range, e.g., 12-15 kilohertz, that coincides with a resonant frequency of the mnicrodispenser  16 —transfer liquid  24  system. 
     In the above description of the invention, the control of the microdispenser  16  was effected by sending a specific number of electrical pulses from the microdispenser electronics  51 , each producing an emitted droplet  26  of transfer liquid  24 . It is also within the scope of the invention to control the microdispenser  16  by monitoring the pressure sensor  14  signal in realtime, and continuing to send electrical pulses to the microdispenser  16  until a desired change in pressure is reached. In this mode of operation, the PC-LPM-16 Multifunction I/O Board that contains the A/D converter  44  is instructed by control logic  42  to send electrical pulses to the microdispenser electronics  51 . Each pulse sent by the Multifimction I/O Board results in one electrical pulse that is sent by the microdispenser electronics  51  to the microdispenser  16 , emitting one droplet  26  of transfer liquid  24 . The control logic  42  monitors the pressure sensor  14  signal as the microdispenser  16  dispense is in progress, and once the desired change is pressure has been attained, the control logic  42  directs the Multifunction I/O Board to stop sending electrical pulses. 
     This mode of operation is employed if a “misfiring” of microdispenser  16  has been detected by control logic  42 . 
     It is also within the scope of the invention for the microvolume liquid handling system  10  to automatically determine (calibrate) the size of the emitted droplets  26  for transfer liquids  24  of varying properties. As heretofore mentioned, emitted droplet  26  size is affected by the properties of the transfer liquid  24 . Therefore, it is desirable to be able to automatically determine emitted droplet  26  size so that the user need only specify the total transfer volume, and the system  10  will internally determine the number of emitted droplets  26  required to satisfy the user request. In the encoded autocalibration algorithm, once the system  10  is primed, an air gap  22  and transfer liquid  24  aspirated, the control logic  42  instructs microdispenser electronics  51  to send a specific number of electrical pulses, e.g., 1000, to the microdispenser  16 . The resulting drop in pressure sensor  14  signal is used by control logic  42  to determine the volume of transfer liquid  24  that was dispensed. This dispensed volume determination is verified by the control logic  42  tracking the volume displaced by the movement of the plunger  34  to restore the system liquid  20  line pressure to the pre-dispense value. 
     The microvolume liquid handling system  10  illustrated is FIG. 1 depicts a single microdispenser  16 , pressure sensor  14 , and pump  12 . It is within the spirit and scope of this invention to include embodiments of microvolume liquid handling systems that have a multiplicity (e.g., 4, 8, 96) of microdispensers  16 , pressure sensors  14 , and pumps  12 . It is also within the spirit and scope of this invention to include embodiments of microvolume liquid handling systems that have a multiplicity of microdispensers  16 , pressure sensors  14 , valves  38 , and one or more pumps  12 .