Piezoelectric-drop-on-demand technology

RELATED APPLICATIONS

This application is a complete application of provisional U.S. application Ser. No. 60/159,701, filed Oct. 15, 1999 and a continuation-in-part of U.S. application Ser. No. 09/489,261, filed Jan. 21, 2000 which is a continuation-in-part of U.S. application Ser. No. 09/056,233, filed Apr. 7, 1998, which is a conventional application of provisional U.S. application Ser. No. 60/041,861, filed Apr. 8, 1997, and provisional U.S. Application No. 60/067,665, filed Dec. 5, 1997, and a continuation-in-part of U.S. application Ser. No. 08/656,455, filed May 31, 1996, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to aspirating and dispensing small volumes of liquids. In particular, it relates to automatic aspirating and dispensing of small volumes of liquids, typically for analytical purposes.

It is possible to accurately aspirate and dispense submicroliter volumes of liquid for analytical applications and to accurately verify the volume of liquid dispensed. The liquid may contain chemically or biologically active substances.

It is also possible to monitor in real time the dispensing of single 100 micron or smaller drops of liquid.

One of the primary factors for successful operation of micromachined or capillary-based piezoelectric-drop-on-demand technology is the ability to keep the dispensers clean.

Multiple technologies exist to clean the outside of the tips by flushing or jetting liquid at the orifice of the dispenser. Dispenser tips can also be immersed in ultrasonic baths to dislodge particles and molecules from the surface of the dispensers.

While these technologies are very effective at cleaning the outside of the dispenser, they do not thoroughly remove deposits adhering to the interior walls. A piezoelectric-drop-on-demand capillary has a very small bore orifice of approximately 5 to 100 &mgr;m and, therefore, cannot be scrubbed by standard mechanical means like a brush or cloth.

Magnetic particles have been associated with various types of separation processes. In recent years magnetic particles have been used to adhere to biological materials to facilitate separation of such materials from the medium in which they are located. One example is found in U.S. Pat. No. 5,895,631 where a high molecular substance is bonded to magnetic particles, which are then attracted to the wall of a container by an external magnetic force. The particles could be removed by releasing the magnetic force which attached the particles to the wall and then flushing them from the container. Similarly, magnetic particles find a new use in the present invention which provides a means for cleaning the interior of the capillary tips used for dispensing 100 micron or smaller drops of liquid.

SUMMARY OF THE INVENTION

In accordance with one aspirating and dispensing apparatus, 1 to 100 micron range drops of liquid are accurately deposited onto various types of locations, typically for analytical purposes.

In another aspect, subnanoliter drops of liquid are dispensed and a pressure change resulting from the droplet ejection is detected. A known volume of a compressible fluid, e.g., a gas such as air, facilitates measuring small changes in system pressure which correlate to the volume of the transfer liquid which has been dispensed.

In accordance with still another aspect, subnanoliter drops of liquid are dispensed, and a pressure change resulting from ejection of a drop of a transfer liquid is detected by an electrical signal which indicates that single drops of liquid are dispensed at millisecond intervals. By eliminating all compressible fluids (gases) from the liquid in the system, the ejection of picoliter size drops can be detected. The dispensed drops are generally in the range of from about 5 to about 500 picoliters, often about 100 to about 500 picoliters.

Subnanoliter droplets of liquid are ejected and the volume of the drops can be measured in real time. Electrical signals indicating transient pressure changes in the transfer liquid upon dispensing liquid drops (in the range of from about 5 to about 500 picoliters, preferably about 100 to about 500 picoliters) can be detected when the liquid in the enclosed volume of the dispenser is 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 a single drop of liquid in the range of from about 5 to about 500 picoliters, preferably about 100 to about 500 picoliters. The pressure change resulting from ejection of such a drop occurs 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.

The invention particularly relates to a means for cleaning the interior of the capillary tubes used to dispense such small droplets, the cleaning being carried out by magnetic particles aspirated into the capillary tubes and moved by a magnet outside the capillary tubes to dislodge deposits adhering to the inner walls.

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 a system for aspirating and dispensing microvolumes of liquid illustrating the first aspirating and dispensing apparatus.

FIG. 2

is a schematic of a positive displacement pump used in the system of FIG.

1

.

FIG. 3

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

FIG. 4

is a block diagram of a system for aspirating and dispensing microvolumes of liquid illustrating the second aspirating and dispensing apparatus.

FIG. 5

illustrates the use of a magnet in combination with magnetic particles to remove deposits adhering to the inner walls of a capillary tip.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention in particular relates to a feature useful in the aspirating and dispensing apparatus described in parent application Ser. No. 09/056,233 and below.

Description of a First Aspirating and Dispensing Apparatus

The system constructed in accordance with the first aspirating and dispensing apparatus of the present invention includes a system liquid and a transfer liquid separated by a known volume of compressible fluid, e.g., a gas such as air (“air gap”). The air gap facilitates measuring small changes in pressure in the system liquid. The change in pressure is proportional to the volume of transfer liquid dispensed. One preferred system liquid is deionized water. As a result of capillary forces, each time a droplet in the microvolume dispensing range is dispensed, the transfer liquid will return to its prior position inside the microdispenser. The specific volume of the air gap will be increased proportionally to the amount of transfer liquid dispensed. The result is a decrease in pressure in the system liquid line which is measured with a highly sensitive piezoresistive pressure sensor. The pressure sensor transmits an electric signal which controls circuitry. The electric signal is converted into a digital form which is indicative of the volume of transfer liquid dispensed. An advantage of the present invention is its insensitivity to the viscosity of the transfer liquid. The pressure change in the system liquid corresponds to the microvolume dispensed, without being dependent on the viscosity of the dispensed liquid.

The first aspirating and dispensing apparatus of the present invention provides 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 process is known as “drop-on-demand.” Specifically, the dispenser of the present invention dispenses drops in the range of from about 5 to about 500 picoliters, preferably from about 100 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.

Referring first to

FIG. 1

, a first microvolume liquid handling system

10

is illustrated, and 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. 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 of about 5 to about 500 picoliters, preferably about 100 to about 500 picoliters per droplet

26

. For example, if one desires to expel a total of

9

nanoliters of transfer liquid

24

, the microdispenser

16

will be directed to expel 20 droplets

26

, each having a volume of 0.45 nanoliters. Droplet

26

size can be altered by varying the magnitude and duration of the electrical signal applied to the microdispenser

16

. Other factors affecting droplet size include: size of the nozzle opening at the bottom of the microdispenser, pressure at the microdispenser inlet, and certain properties of the transfer liquid.

Referring now to

FIGS. 1 and 2

, in one preferred embodiment, the positive displacement pump

12

is an XL 3000 Modular Digital Pump, manufactured by Cavro Scientific Instruments, Inc., Sunnyvale, Calif. The positive displacement pump

12

includes stepper motor

28

, 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 full

34

stroke. In one preferred embodiment, the stepper motor

28

is directed to make 12,000 steps per plunger

34

full 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 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 the dilution of certain system liquids. 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, the MultiPROBE CR10100, manufactured by Packard Instrument Company. 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

. This causes the valve

38

to rotate (by means of stepper motor

29

), 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 uppermost position (Position 1 in

FIG. 2

) in 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 lowermost position (Position 2 in

FIG. 2

) in the tube

32

and 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

that is, in turn, 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, manufactured 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 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 converter 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

exist 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 microdispenser, 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

detects fluctuations in pressure that occur with priming the microvolume liquid handling system

10

, aspirating transfer liquid

24

with a pump

12

, dispensing droplets

26

with the microdispenser

16

, and washing of the microdispenser

16

with a pump

12

. In one preferred embodiment, the pressure sensor

14

is a piezoresistive pressure sensor, part number 26PCDFG6G, manufactured by Microswitch, Inc., a division of Honeywell, Inc., Freeport, Ill. Also included with the pressure sensor

14

in the block diagram in

FIG. 1

is electrical circuitry which amplifies 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 used by the control logic

42

. For example, when the microvolume liquid handling system

10

is being primed, the pressure sensor

14

sends electrical signals which are analyzed by the control logic

42

to determine whether they indicate 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 the 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). Contact of the microdispenser

16

with the surface of the transfer liquid

24

is determined by a capacitive liquid level sensing 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 the transfer liquid

24

surface, a signal is sent to the robotic system

58

through electrical wire

53

to stop the downward motion.

The control logic

42

instructs the pump

12

to move the plunger

34

down to aspirate the transfer liquid

24

into the microdispenser

16

. To ensure that the transfer liquid is successfully drawn into the microdispenser, the pressure signal is monitored by control logic. If a problem, such as an abnormal drop in pressure due to partial or total blockage of the microdispenser is detected, the control logic

42

will send a stop movement command to the pump

12

. The control logic

42

will then proceed with an encoded recovery algorithm. Note that the transfer liquid

24

can be drawn into the microvolume liquid handling system

10

up to the pressure sensor

14

without the 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 microtiter plate or a wafer.

In one preferred embodiment, the microdispenser

16

is the MD-K-130 Microdispenser Head, manufactured by Microdrop, GmbH, 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

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. The microdispenser electronics

51

control the magnitude and duration of the analog voltage pulses, as well as 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 highly accelerated droplet

26

of transfer liquid

24

is emitted. 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, thus allowing extremely small (e.g., 5 picoliter) droplets

26

to be expelled from the nozzle. Use of the microdispenser

16

to propel droplets

26

out of the nozzle circumvents problems encountered in the liquid transfer technique referred to “touchoff.” In the touchoff technique, a droplet

26

is held at the end of a nozzle and is deposited onto a target surface by bringing that droplet

26

into contact with the target surface while it is still suspended from the microdispenser

16

. Such a contact process is susceptible to unacceptable volume deviations as a result of surface tension, viscosity and wetting properties of the microdispenser

16

and the target surface. 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

, located between the piezoelectric transducer

60

and the nozzle

63

, is plated with a conductive material, typically platinum or gold. The use of this material 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

, thus 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

. Since the transfer liquid-air gap system liquid column terminates at a closed end in the positive displacement pump

12

, however, there is a corresponding drop in the system liquid

20

line pressure as the air gap

22

is expanded. This may be seen in

FIG. 4

of parent application Ser. No. 09/056,233. 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

is proportional to the volume dispensed. Thus, from the pressure change measured by the pressure sensor

14

, the control logic determines the volume of transfer liquid

24

that was dispensed. In one preferred embodiment of the present invention, depending on the properties of the transfer liquid, it is preferable that the drop in pressure not exceed approximately 30 to 40 millibars below ambient pressure. 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., the difference between the ambient pressure acting on the nozzle

63

and the pressure at the capillary inlet

65

) 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 verify 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

that monitors 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. Because the control logic

42

tracks the displaced volume, the plunger

34

moves (20.83 nanoliters per stepper motor

28

step) and a second confirmation of the volume dispensed is made, thus adding robustness to the system. After a second dispensing verification, the system liquid

20

line pressure is now at the correct value for the next dispensing action if a multi-dispense sequence has been specified.

Once the transfer liquid

24

dispensing has been completed, the control logic

42

causes the robotic system

58

to position the microdispenser

16

over the wash station. The control logic

42

then directs pump

12

and robotic system

58

in a wash protocol that disposes of any transfer liquid

24

left in the microdispenser

16

. This protocol also results in washes to the internal surface of the 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 protocol is designed to minimize cross-contamination of different transfer liquids

24

used during different dispensing sessions. Towards 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 using the control logic

42

to direct the microdispenser electronics

51

to send electrical pulses to the microdispenser at a frequency in the range of from about 1 to about 20 Khz (the preferred resonant frequency of the microdispenser

16

is believed to be approximately 12 kilohertz). The resonant frequency of the microdispenser coincides with the 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. 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 the above-described frequencies. Cleaning with high frequency pulsing is more efficient at 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 in the microdispenser

16

compared to cleaning without such pulsing.

Pulsing of the microdispenser

16

is also 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

. In biochemical applications, one widely used matter added to the transfer liquid

24

is polystyrene spheres. These spheres typically range from 1 micron to over 30 microns, 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 glutinous coatings, can cause the spheres to adhere to the nozzle

63

. It has been found that if the piezoelectric transducer

60

is excited at high frequency while the microdispenser

16

is being loaded (i.e., transfer liquid

24

is being aspirated into the microdispenser

16

), clogging may be prevented or minimized. Thus, high frequency pulsing of the microdispenser

16

prevents or diminishes 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

, the possibility of clogging occurs. 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

. 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 to this cleaning strategy is continuous instrument operation without the delays associated with alternate cleaning procedures. In short, system downtime is reduced, making the microvolume liquid handling system

10

more efficient.

In certain applications, such as those described above, the liquid being dispensed adheres to the inner walls of the capillary tip and, despite the pulsing with the piezoelectric transducer, additional cleaning is required. It has been found that abrasive magnetic particles can be used to clean the capillary tips by manipulating them with an external magnet.

In the above description of the invention, the control of the microdispenser

16

occurs via electrical pulses from the microdispenser electronics

51

, with each pulse resulting in 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 real time, 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 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 dispensing is in progress. Once the desired change in pressure has been attained, the control logic

42

directs the Multifunction I/O Board to discontinue 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 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 to satisfy the user requirements. In the encoded autocalibration algorithm, once the system

10

is primed, an air gap

22

and transfer liquid

24

are aspirated, and the control logic

42

instructs the 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 the control logic

42

to determine the volume of transfer liquid

24

that was dispensed. The control logic verifies the volume of liquid dispersed by tracking the volume displaced by the movement of the plunger

34

. The system subsequently restores the liquid line pressure to the pre-dispense value.

The microvolume liquid handling system

10

illustrated in

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

.

Description of a Second Aspirating and Dispensing Apparatus

In

FIG. 4

, another aspirating and dispensing apparatus

210

is shown. This embodiment, which is preferred when the number of microdispensers employed is equal to or greater than eight, also realizes the aforementioned objectives. The second apparatus is similar to the first shown in

FIG. 1

, 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 and controlling system liquid pressure. This embodiment also employs a plurality of flow sensors for detecting liquid flow, as well as pressure in the system liquid which is present in the connecting tubing that is coupled to each microdispenser. It also employs a 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 apparatus, 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

utilized in the first apparatus. Note that first and second 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 second 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 the use of 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 an 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 adjusts the pressure of the system liquid

20

and, correspondingly, the pressure of the transfer liquid

24

via an electrical conductor. 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 corresponds to a different phase of operation of the microvolume liquid handling system

210

. The three different pressure levels include a positive pressure, a high negative pressure, and a low negative pressure. Prior to dispensing, positive pressure is used to clean the microdispenser. High frequency pulsing of the microdispensers

212

is also employed in the manner described above. After the microdispensers

212

are relatively clean, the high negative pressure levels (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 levels (roughly −15 millibars gauge) are 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. 4

, with one section

236

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

to determine the amount of liquid that is being aspirated into each microdispenser. The flow sensor

244

detects the flow of system liquid

20

into or out of each microdispenser

212

. The flow sensors

244

are each connected to the system controller

224

through an electrical conductor

248

. The electrical conductor

248

carries electrical signals from each flow sensor

244

, indicating not only the amount of liquid flow, but also the pressure in each flow sensor. The flow sensors

244

are microfabricated. This is advantageous since the sensors are small and fit easily into the microvolume liquid handling system

210

. An example of the flow sensors

244

is described in IEEE Proceedings, MEMS 1995, Publication No. 0-7803-2503-6, entitled, “A Differential Pressure Liquid Flow, Sensor For Flow Regulation and Dosing Systems,” by M. Boillat et al., hereby incorporated by reference.

The distribution tube

234

, which is physically connected to the microdispensers

212

, is attached to a three axis robot

238

. As in the first preferred embodiment, the microdispensers are relocated to positions above different microtiter plates, wells or wafers. After the desired number of droplets has been dispensed, the robot

238

moves the microdispensers

212

to the next set of wells or wafers for further dispensing. The dispensing heads can be stationary and the robotic system can be used to locate the source and destination vessels.

It has been discovered that the ejection of individual drops of a transfer liquid in the volume range of about 100 to about 500 picoliters can be detected using the system of the present invention with a pressure detector. In order to detect dispensing of such drops, the transfer and system liquids must be substantially free of compressible gases, such as air. As used herein, the term “substantially free of compressible gas” means that the level of compressible gas, if any, is low enough to allow the detection of a drop of liquid being ejected from the system. It has been discovered that as the amount of compressible gas in the system increases, the ability to detect dispensing of the drop decreases until, at a certain level of compressible gas, the system cannot detect dispensing of a drop of the transfer liquid.

In accordance with one embodiment of the present invention, the volume from the dispensing nozzle, which holds the transfer liquid to the valve (

242

in FIG.

4

), is substantially free of compressible gas and is entirely enclosed. It has been discovered that in this preferred embodiment of the present invention, drops can be ejected from the closed volume until the pressure in the fluid is reduced to about −45 millibars gauge. At about −45 millibars gauge the vacuum interferes with the ejection of the drops.

In accordance with another embodiment of the present invention, the volume from the dispensing nozzle to the reservoir of system liquid is substantially free of compressible fluid (gas). It has been discovered that upon dispensing a drop of liquid, the system of this embodiment can detect a pressure change in the system liquid resulting from such drop being dispensed. The pressure change is transient. As the transfer liquid flows into the volume adjacent to the nozzle, effectively replacing the ejected drop volume, the pressure rises to the level prior to the dispensing of the drop. It has been discovered that for dispensing drops in the size range of from about 100 to about 500 picoliters, the time required for the pressure to reach the original level can be in a range of from about 5 to about 10 milliseconds. This time period can be controlled by selecting the size and confirmation of the orifice located between the volume that is adjacent to the nozzle and the reservoir. It has been determined that purging the air out of the system with a fluid (gas) that has a high solubility coefficient with respect to the system liquid has greatly reduced the residual compressible fluid (gas) in the system after priming with system liquid. Once the system is primed, keeping compressible fluids (e.g., air) out of the system is facilitated by degassing the system liquid, pressurizing the system liquid reservoir with an inert gas, utilizing low permeability tubing, and also degassing system liquid in-line. To aid in elimination of air bubbles, carbon dioxide purging can be employed as described in IEEE Proceedings, MEMS 1995, Publication No. 0-7803-2503-6, entitled “Carbon Dioxide Priming Of Micro Liquid Systems,” by R. Zengerle et al.

An example of the ability of the system to dispense single drops is provided in parent application Ser. No. 09/056,233, and illustrated in

FIGS. 8-11

.

In accordance with another aspect of the present invention, several methods have been developed to minimize the amount of transfer liquid that needs to be aspirated into the dispenser. In the system of the present invention, which is capable of monitoring the ejection of single drops, the dispensing chamber has to be free of compressible fluids (gas) in order for the drops to be ejected. This requires that the chamber from the nozzle (

63

in

FIG. 3

) to the top of the piezoelectric transducer (

60

in

FIG. 3

) be filled with liquid. This volume is often large in comparison to the volume of transfer liquid to be dispensed.

In accordance with one method, the system liquid and the transfer liquid are not separated from each other by an air gap, as shown in FIG.

1

. Instead, the two liquids are separated by a liquid which is immiscible with either or both the transfer liquid and the system liquid.

In accordance with another method, to minimize the required aspirate volume of transfer liquid, system liquid is used to fill the dispenser before aspiration of the transfer liquid begins. It has been discovered that, as the transfer liquid is aspirated, the system liquid mixes with the transfer liquid at the interface slowly enough to allow dispensing of a large percentage of the transfer liquid without observing a dilution of the transfer liquid with the system liquid.

In embodiments which do not require use of a separate system liquid, a single liquid can be used to serve as both the system liquid and the transfer liquid.

In accordance with a further aspect of the present invention, the pressure in the dispenser (such as in dispenser

212

of

FIG. 4

) decreases as a result of a reduction in the system liquid reservoir (

214

in

FIG. 4

) pressure. The valve (

242

in

FIG. 4

) is closed, and then the nozzle of the dispensing unit can be immersed in the transfer liquid to aspirate a small quantity of the transfer liquid into the dispenser. For example, when gauge pressure in the dispenser reaches −30 millibars, submersing the nozzle in the transfer liquid may draw a sufficient amount of liquid to increase the gauge pressure to −15 millibars. It should be noted that the dispenser does not aspirate air unless the surface tension in the nozzle is exceeded by the negative gauge pressure. In the system of the preferred embodiment using dimethyl sulfoxide, the negative gauge pressure of 45 millibars does not produce air aspiration into the nozzle.

The systems described can automatically detect when the microdispenser orifice enters into a liquid and when it is withdrawn.

A pressure-based liquid detection function has been developed for the embodiments shown in

FIGS. 1 and 4

. This function can be used to detect when one or more micro dispensers is immersed in or withdrawn from liquid. This determination is made based on a pressure change which occurs when the microdispensers are immersed in or withdrawn from liquid. This pressure change is measured by monitoring the pressure transducer (

14

in

FIG. 1

) or flow sensors (

244

in FIG.

4

). This test is performed independently for each system microdispenser.

The liquid determination process can be divided into three distinct stages.

1. Predelay

Upon receipt of a “liquid level sense” command, the algorithm allows for a user-specified predelay to be performed. The duration of the delay allows the completion of an external event (i.e., the movement of the head to an aspiration source) to occur before the software begins to look for the pressure change of an air/liquid transition. Certain external events may result in a false positive if these events trigger a pressure change. This function allows the system to identify any spurious pressure change.

In the event that the predelay is zero, the software will begin monitoring the pressure immediately upon receipt of the “liquid level sense” command. This can also be applied in systems where the microdispensers are stationary and the robotic system moves the source, or aspiration vessel.

2. Baseline Establishment

Once the predelay has expired, a baseline pressure value is established from the average of multiple readings. This baseline pressure value will then be compared to subsequent pressure readings to determine if they differ enough to indicate an air-liquid transition.

3. Liquid Detect

The last stage is utilized to compare the established baseline pressure value with the current pressure values. The current pressure value is a rolling average. This ensures that a single spurious point will not result in an incorrect liquid detection event. During this stage, the pressure is read periodically. The oldest pressure value is then removed, the newest pressure value added, and a new average calculated. This average is then compared with the baseline which was established in the previous stage. The difference between these values is assessed via a user-specified threshold value. If the magnitude of the difference is greater than the threshold, then the algorithm will conclude that a liquid detect event has occurred and will set the liquid detected states to the control logic. The same test is performed independently for each dispenser.

The algorithm will continue to monitor the system for liquid detection events until a user-specified detection duration has expired. If no pressure transition of the specified magnitude occurs during this duration, the software will notify the control logic that no air-liquid transition has occurred for that particular dispenser.

The user-specified threshold value, in units of millibar, is used to refine the liquid detection process. If true air-liquid transitions are occurring, but are not being identified, then the threshold value can be decreased, thus enhancing detection sensitivity. If false liquid-detection determinations are being made as a result of random pressure fluctuations, the threshold value can be increased, thus diminishing detection sensitivity. The pressure threshold has a positive or negative value associated with it, thus enabling the user to activate the liquid detection function when the microdispensers are either immersed in or withdrawn from liquid.

Cleaning With Magnetic Particles

As previously discussed, capillary tubes may be pulsed at high frequency to prevent or eliminate clogging. When more complete cleaning is required, abrasive magnetic particles may be aspirated into the tip and moved by an external magnet to remove deposits on the inner wall of the capillary. The concept is illustrated in FIG.

5

. Magnetic particles, for example those used to bind and extract DNA, RNA, and the like from solutions, may be used to clean the interior walls of capillary tubes as described above. Although such particles are convenient, since they are commercially available and are capable of binding the deposits, other types of fine magnetic particles may be used. The particles must be small enough to pass through the capillary tip easily and are typically about 0.2 to 4 &mgr;m in diameter. Also, they must have sufficient interaction with magnets outside the capillary to be moved against the interior wall of the capillary with sufficient force to dislodge adhering deposits.

FIG. 5

illustrates schematically the interaction of a ring magnet surrounding a capillary tip. Either the magnetic is moved to move the particles or alternatively, the capillary tip may be moved as was done in the following example. The piezoelectric transducer may be used to assist removing the deposits and magnetic particles while flushing the capillary tube.

EXAMPLE

Nucleotide quality magnetic particles obtained from Promega were added to a solution of CY3-labeled oligonucleotide material having a concentration of 0.1 &mgr;g/&mgr;L in a 50 m molar borate buffer.

A piezoelectrically-operated capillary tip was flushed for 4 seconds over a standard washbowl, then 10 &mgr;L of the magnetic particle containing solution was aspirated into the capillary tube. After which, the capillary tube was moved up and down in another washbowl containing a ring magnet having an internal diameter of 2 mm and external diameter of 7 mm, while a wash fluid was flushed through the capillary tube to remove dislodged deposits. Finally, the capillary tube was moved to the original washbowl and the remaining fluid containing the magnetic particles binding the oligonucleotide material was expelled from the capillary tip.

While the present invention has been described with reference to one or more particular embodiments, those skilled in the art will recognize that many changes may be made thereto without departing from the spirit and scope of the present invention. Each of these embodiments, and obvious variations thereof, is contemplated as falling within the scope of the claimed invention, which is set forth in the following claims.