Microvolume liquid handling system

RELATED APPLICATIONS

This patent application is a continuation-in-part application of a U.S. patent application Ser. No. 08/656,455, filed May 31, 1996, now abandoned a pending provisional U.S. patent application Ser. No. 60/041,861 filed Apr. 8, 1997, and a pending provisional U.S. patent application Ser. No. 60/067,665 filed Dec. 5, 1997.

FIELD OF THE INVENTION

The present invention relates to an apparatus and process for controlling, dispensing and measuring small quantities of liquids. More specifically, the present invention senses pressure changes to ascertain and confirm the volume dispensed liquids and proper system functioning. In particular, the present invention relates to aspirating and dispensing picoliter range droplets of liquid.

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 liquids containing chemically or biologically active substances for commercial or experimental use. Accuracy and precision in the amount of liquid 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 liquids 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 liquids 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. Heavy use of the nozzle promotes undesirable clogging by materials in the liquid being dispensed. 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 prevent or minimize clogging, 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 liquid can also be caused by other factors, such as air or other compressible gases being trapped 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”).

Over time it may be necessary to aspirate a variety of different liquid mixtures or solutions into the microvolume dispenser in order to dispense those liquids. Because each liquid may contaminate the microvolume dispenser with regard to a later-used liquid it is desirable to thoroughly clean a microdispenser when liquids are changed. Even when liquids are not changed, cleaning is necessary to prevent buildup of materials inside the microvolume dispenser. Unfortunately, using a pump alone to flush out the microvolume dispenser is not always 100% effective. Therefore, it would be desirable to be able to easily and thoroughly clean the microvolume dispenser from time to time.

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. For example, it is desirable to ensure that the transfer liquid is accurately deposited at its target surface. Because industry requires rapid dispensing of microvolume amounts of transfer liquid, it is also desirable to be able to ascertain transfer liquid volume dispensed, and to be able to detect and recover from dispensing problems in realtime.

One 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.

A further 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.

Another object of the present invention to provide a microvolume liquid handling system which can transfer microvolume quantities of liquids containing chemically or biologically active substances.

A further object of the present invention to provide a microvolume liquid handling system that prevents or minimizes clogging.

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.

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.

A further object of the present invention to minimize the amount of transfer liquid that needs to be aspirated into the dispenser.

A still further object of the present invention to automatically detect when the dispenser tip enters and leaves the surface of the transfer liquid and/or wash liquid.

A still further object of the present invention is to provide for a real time detection of dispensing single drops of the transfer liquid.

Other objects and advantages of the present invention will be apparent to those skilled in the art upon studying of this application.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, a system of the present invention detects a pressure change resulting from ejection of a drop of a transfer liquid and generates an electrical signal indicating single drops of transfer liquid being dispersed in intervals measured by milliseconds. The dispersed drops being detected by the system can be in the range from about 5 picoliters to about 500 picoliters, preferably about 100 to about 500 picoliters. It has been discovered that by eliminating substantially all compressible fluids (gases) in the enclosed volume communicating with the ejection nozzle and containing the transfer liquid, the ejection of picoliter size drops can be detected by the present invention.

In accordance with another aspect of the present invention, it has been discovered that electrical signals indicating transient pressure changes in the transfer liquid upon dispensing of liquid drops in the range from about 5 picoliters to about 500 picoliters, preferably about 100 to about 500 picoliters can be detected even when the liquid in the enclosed volume of the dispenser is not fully enclosed, but is instead connected to a liquid reservoir. As long as substantially all compressible fluids (gases) are kept out of the dispensing conduit which communicates through a restricted passage to the liquid reservoir, the pressure sensor of the system of the present invention can detect dispensing of a single drop of liquid, having a size range from about 5 picoliters to about 500 picoliters, preferably 100 picoliters to about 500 picoliters. The pressure change resulting from ejection of such a drop returns to the pre-ejection pressure level in a time period long enough for the pressure change to be detectable but short enough to complete the cycle before the next drop is ejected. In the preferred embodiment, the drops were ejected within 10 milliseconds of each other and depending on the operating conditions the pressure returned to the normal level in the time range from about 5 to about 10 milliseconds.

In accordance with another aspect of the present invention clogging is prevented or minimized by pulsing the piezoelectric transducer at frequencies in the range from about 1 KHz to about 20 Khz. If the microdispenser is determined to be clogged by the control logic, frequencies close to the resonant frequency of the microdispenser are generally used, generally about 12 KHz. The piezoelectric transducer can also be pulsed at or near the resonant frequencies when the microdispenser is being cleaned. The resonant vibrations of the microdispenser during cleaning result in a cleaner microdispenser interior than without vibration. Because the same transducer is used to prevent clogging, to break up existing clogs and to clean the microdispenser, greater efficiencies are achieved than previously possible.

In accordance with still another aspect of the present invention enables the microdispensers to be positioned with a high degree of accuracy with regard to wells of a microtitre plate. Visible or infrared light is transmitted through a transparent bottom half of a microtitre plate containing wells organized in rows and columns. Light does not pass through the opaque top half of the microtitre plate. When a particular microdispenser is moved from a position above the opaque top half of the microtitre plate to a position above the transparent bottom half of the microtitre plate, light passes through the glass capillary in the microdispenser where it is detected by a photo detector in optical contact with the glass capillary. The photo detector generates electronic signals corresponding to the amount of light received. The signals from the photo detector are coupled to a computer which uses the signals to help locate and verify the position of the microdispenser.

In accordance with another aspect of the present invention, the liquid surface in a vessel is detected and the microdispenser orifice is located based on the change of the pressure which occurs when the orifice of the microdispenser is in communication with a liquid reservoir.

Other aspects of the present invention will become apparent to those skilled in the art upon studying this disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

is a block diagram of the a microvolume liquid handling system illustrating the first embodiment of the present invention.

FIG. 2

is a schematic of a positive displacement pump illustrating an aspect of the first embodiment of the present invention.

FIG. 3

is side plan view of a microdispenser including a piezoelectric transducer.

FIG. 4

is a graph depicting the system pressure measured during dispensing of microvolume of a liquid using a microdispenser of the present invention.

FIG. 5

is an exploded perspective view of two halves of a microtitre plate prior to being joined, as used with the present invention.

FIG. 6

is a sectional side plan view showing the two halves of the microtitre plate after having been joined in accordance with the present invention.

FIG. 7

is a block diagram of the a microvolume liquid handling system illustrating the second embodiment of the present invention.

FIG. 8

is a plot of the pressure (in millibars) detected in eight dispenser heads as a function of time while dispensing drops of dimethyl sulfoxide at 100 drops per second with valves to the reservoir shown in

FIG. 7

in an open position.

FIG. 9

is a plot the pressure (in millibars) detected in two of the dispensers shown in

FIG. 8

, on an expanded pressure scale.

FIG. 10

is a plot of the pressure (in millibars) detected in eight dispenser heads as a function of time while dispensing a single drop of dimethyl sulfoxide with the valves to the reservoir shown in

FIG. 7

in an open position.

FIG. 11

is a plot showing the pressure (in millibars) detected in two of the dispensers shown in

FIG. 10

, on an expanded pressure scale.

FIG. 12

is a block diagram of the program logic for operating pressure-based liquid detect feature of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The system of the present invention possesses unique capabilities in microvolume liquid handling. Surprisingly, it has been discovered that sub-nanoliter droplets of liquid can be dispensed with real time verification. Dispensing of a single sub-nanoliter drop can be detected in real time. As the result of dispensing the liquid in sub-nanoliter droplets, the dispensed volume can be precisely controlled. The dispenser of the present invention can automatically detect the liquid surface of the transfer liquid, automatically aspirate, analyze desired volume of the transfer liquid, dispense the transfer liquid without contacting the destination vessel or its contents, and automatically wash off the transfer liquid from dispensing system after each transfer. 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 system of the present invention is pulsated at high frequency to prevent or eliminate clogging. Immiscible liquids between the transfer liquid and the system liquid reduces the required amount of transfer liquid needed for dispensing.

Description of the First Preferred Embodiment

The system constructed in accordance with the first preferred embodiment of the present invention includes a system liquid and a transfer liquid separated by a known volume of gas, such as air, (“air gap”) the air gap 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 one preferred 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'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 viscosity of the dispensed liquid.

A first preferred embodiment 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, this is known as ‘drop-on-demand’. Specifically, the dispenser of the present invention disperses drops in the range from about 5 picoliters to about 500 picoliters, preferably from about 100 picoliters to about 500 picoliters.

To provide the functionality of an automated liquid handling system, the microdispensers in all preferred embodiments are mounted onto a 3-axis robotic system that is used to position the microdispensers at specific locations required to execute the desired liquid transfer protocol.

Turning now to the drawings and referring first to

FIG. 1

, a first embodiment of 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 is provided. Then, an amount of transfer liquid

24

is drawn into the microdispenser

16

in a manner described below. The transfer liquid

24

can contain one or more biologically or chemically active substances of interest. Preferably, 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 generally in the range 5 to 500 picoliters, preferably 100 to 500 picoliters per droplet

26

. 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

, each having a volume of 0.45 nanoliters. 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 liquid-tight 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 full 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 liquid 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 preferred 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 liquid 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 Norderstedt, 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 eliminates 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 specifying 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 further 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 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 liquid 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 use a high frequency pulsing of the transducer

60

to facilitate washing 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 range from about 1 to 20 Khz, preferably 12-15 kilohertz (the preferred resonant frequency of the microdispenser

16

is believed to be approximately 12 kilohertz), that coincides with a resonant frequency of the microdispenser

16

—transfer liquid

24

system. Pulsing the piezoelectric transducer

60

at the above frequencies causes the interior surfaces of the glass capillary

62

to vibrate vigorously. In both the first and third embodiments, system liquid

20

or a special cleaning and/or neutralizing liquid is used to flush out the microdispenser

16

while the piezoelectric transducer

60

is activated at frequencies. Cleaning with pulsing at high frequencies has the effect of far more efficiently dislodging and eliminating matter adhering to the microdispenser

16

. For example, it has been shown in a number of test cases that such cleaning caused a 200% to 500% improvement (depending on the contaminant) in the reduction of residual matter left in the microdispenser

16

as compared to cleaning without such pulsing.

Pulsing of the microdispenser

16

also is used to prevent, minimize or alleviate clogging of the nozzle of the microdispenser. For example, when transfer liquid is being aspirated into the microdispenser

16

it must pass through the relatively narrow nozzle

63

in the glass capillary

62

. Matter in the transfer liquid

24

often comes into contact with the nozzle's

63

surfaces permitting the matter to adhere to the nozzle

63

, depending on the nature of the contact. In biochemical applications, one widely used matter added to the transfer liquid

24

is polystyrene spheres. These spheres typically range from 1 &mgr;M to over 30 &mgr;M and may be uncoated or coated with magnetic ferrites, antigens or other materials. The relatively large size of the polystyrene spheres with regard to nozzle

63

diameter, in combination with their sometimes sticky coatings, can cause the spheres to adhere to the nozzle

63

. It has been discovered that if the piezoelectric transducer

60

is excited at high frequency of the microdispenser

16

while the microdispenser

16

is being loaded (i.e. transfer liquid

24

is being aspirated in to the microdispenser

16

) that clogging is prevented or less likely to occur. Thus, high frequency pulsing of the microdispenser

16

works to prevent or diminish clogging of the nozzle

63

by materials in the transfer liquid

24

.

Anytime a transfer liquid

24

containing dissolved or suspended materials passes through the nozzle

63

there is a possibility of clogging. Accordingly, not only is clogging a problem during aspiration of transfer liquid

24

into the microdispenser

16

as described above, but it is also a problem when transfer liquid is dispensed from the high frequency pulsing of the microdispenser

16

between droplet dispensing by the piezoelectric transducer can reduce buildup of materials adhering to the nozzle

63

and thus prevent clogging in some instances. Even if substantial clogging does occur, high frequency pulsing of the microdispenser

16

by the piezoelectric transducer

60

will substantially clear the clogging materials from the nozzle

63

. The key advantage here is that by preventing or eliminating clogging of the nozzle

63

, the microvolume liquid handling system

10

can continue operation without resort to extraordinary cleaning procedures and the delays associated with those procedures. In short, system downtime is reduced, thereby making the microvolume liquid handling system

10

more efficient.

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 Multifunction 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

.

Second Preferred Embodiment

Turning now to

FIGS. 5 and 6

, one application for drop-on-demand microvolume liquid dispensing is to deposit precise amounts of transfer liquid

24

into an array of wells in a microtitre plate

110

, which is described in U.S. Pat. No. 5,457,527, hereby incorporated by reference. The microtitre plate

110

is formed from two molded plastic plates

111

and

112

. The upper plate

111

forms the side walls

113

of the multiple wells of the microtitre plate, and in the illustrative example, the wells are arranged in an 8×12 matrix, although matrices with other dimensions also work with the present invention. The bottom plate

112

forms the bottom walls

114

of the matrix web, and is attached to the lower surface of to the lower surface of the upper plate by fusing the two plates together. The upper plate

111

is formed from an opaque polymeric material so that light cannot be transmitted through. In contrast to the upper plate

111

, the lower plate

112

is formed of a transparent polymeric material so that it forms a transparent bottom wall

114

for each sample well. This permits viewing of sample material through the bottom wall

114

, and also permits light emissions to be measured through the bottom wall. The transparent bottom walls

114

may also be used to expose the sample to light from an external excitation source, while leaving the tops of the wells unobstructed for maximum detection area.

In part because the present microvolume liquid dispensing system

10

can precisely dispense extremely small quantities of liquid, it is possible to utilize microtitre arrays

110

of correspondingly reduced dimensions. The difficulty of positioning the nozzle

63

directly over each well increases as the well diameter approaches the one millimeter range. In the case of a well diameter of one millimeter, it is desirable to position the nozzle

63

within 150 micrometers (“&mgr;M”) of the center of the well to permit accurate droplet shooting. The present invention utilizes a transparent bottom portion

112

of the microtitre plate array

110

, which allows visible and infrared light to pass through the bottom of the microtitre array

110

into the well formed by the opaque side walls

113

of the microtitre plate array

111

and the transparent bottom walls

114

of the transparent bottom array

112

. In one embodiment infrared light is passed through the transparent bottom section

112

of the microtitre plate array

110

onto the glass capillary

62

of the microdispenser

16

. The light received at the microdispenser

16

is passed through the glass capillary

62

to an appropriate infrared detector (not shown) mounted on the glass capillary

62

. The infrared light source, in combination with the narrow well structure, provides a narrow beam of infrared light directed upward through each well, but not through an opaque material between the wells. As the microdispenser is moved from one well to another it encounters a relatively dark zone indicating the dispenser is between wells, followed by a relatively bright zone indicating the edge of the next well is directly below. The positioning robot then uses these cues to reach and verify the position of the microdispenser.

In another preferred embodiment, visible light is used in place of infrared light as described above. For example, any visible wavelength of light can be used if the wells are devoid of liquid, or have clear liquids and a matching detector is used in place of the infrared detector. In the case where a turbid or cloudy liquid is present in the wells, a greenish light at 300 nM can be passed through the microtitre plate

110

to the turbid liquid. A cryptate compound added to the liquid present in the well fluoresces in response to excitation by the greenish light. Cryptate fluoresces at approximately 620 and 650 nM, corresponding to red light. A detector that detects those red wavelengths is used in place of the infrared detector.

Third Preferred Embodiment

Turning now to

FIG. 7

, another preferred embodiment of the microvolume liquid handling system

210

is shown. This preferred embodiment of the microvolume liquid handling system, which is more preferred than the first preferred embodiment when the number of microdispensers employed is equal to or greater than eight, also realizes the foregoing objectives. The third preferred embodiment is similar to the first preferred embodiment, except that the positive displacement pump (which includes a valve as described below), the stepper motor, and the piezoresistive pressure sensor are replaced with a pressure control system for supplying system liquid and controlling system liquid pressure, a plurality of flow sensors for detecting liquid flow as well as pressure in the system liquid present in connecting tubing coupled to each microdispenser, and plurality of valves (such as solenoid or microfabricated valves), each valve coupling each microdispenser to a system reservoir in the pressure control system. In this preferred embodiment, a system liquid reservoir

214

is used to supply system liquid

20

to all the microdispensers

212

, thus eliminating the separate pump and pressure sensor for each microdispenser

212

in the first preferred embodiment. Note that first and third preferred embodiments are otherwise identical in structure and operation except as described herein. The precise number of microdispensers employed is a function of the user's dispensing requirements.

With regard to the third preferred embodiment, the system liquid reservoir

214

receives system liquid

20

, typically deionized water or dimethyl sulfoxide (DMSO), through an intake tube

216

which contains a cap (not separately shown). The cap on the intake tube

216

is removed to enable the sealed system liquid reservoir

214

to receive system liquid

20

when the cap is off and seals the system liquid reservoir

214

shut when the cap is on so that the system liquid reservoir

214

can be maintained at a desired pressure. Pressure in the system liquid reservoir

214

is maintained by a pressure control system

218

, through pressure control tubing

220

. The pressure control system

218

includes an electrically controlled pump capable of accurately increasing or decreasing pressure in the system liquid reservoir

214

. A pressure sensor

222

mounted on the system liquid reservoir

214

senses pressure in the system liquid reservoir

214

and transmits an electrical signal indicative of that pressure to a system controller

224

through electrical conductor

226

. The system controller

224

contains a digital signal processor board and other electronics(not shown) which enable monitoring of various electrical signals, execution of control software code, and control of the microvolume liquid handling system

210

. The system controller

224

electrically controls the pressure control system

218

through an electrical conductor

228

to adjust the pressure of the system liquid

20

, and correspondingly, the pressure of the transfer liquid

24

. A pressure relief valve

230

is mounted on the system liquid reservoir

214

. The pressure relief valve

230

releases pressure from the system liquid reservoir

214

when the pressure exceeds a predetermined safety threshold. In one embodiment, the pressure relief valve

230

can also be opened by the system controller

224

which is connected to the pressure relief valve

230

by a wire

232

.

During operations, the system controller

224

directs the pressure control system

218

to maintain one of three different pressure levels in the system reservoir

214

with regard to ambient atmospheric pressure. Each of the three pressure levels correspond to a different phase of operation of the microvolume liquid handling system

210

. The three different pressure levels are a positive pressure, a high negative pressure and a low negative pressure. Prior to dispensing, the positive pressure level is used for cleaning in order to wash the microdispenser free of any foreign matter in combination with high frequency pulsing of the microdispensers

212

in the manner described above. After the microdispensers

212

are relatively clean, the high negative pressure level, roughly 200 millibars less than the ambient atmospheric pressure, is used to aspirate transfer liquid

24

into the microdispensers

212

. Once the transfer liquid

24

has been aspirated into the microdispensers

212

, the low negative pressure level, roughly −15 millibars, is used to supply back pressure to the transfer liquid

24

in the microdispensers

212

such that as droplets are dispensed, no additional transfer liquid

24

leaves the microdispensers

212

.

System liquid

20

in the system reservoir

214

is coupled to the microdispensers

212

through a distribution tube

234

that splits into a plurality of sections

236

as shown in

FIG. 7

, one section

236

is connected to each microdispenser

212

. Attached to each of the distribution tube sections

236

are solenoid valves

242

and flow sensors

244

. The system controller

224

sends electrical signals through an electrical connection

246

to control the valves

242

. A flow sensor

244

is attached to each distribution tube section

236