Patent Application
20090054266
The invention generally relates to techniques for assaying small volumes of liquid, and more specifically to physical transfer of a small volume into a storage medium.
Techniques are rapidly developing for parallel performance of a large number of chemical and biological assays and synthesis operations. One approach uses a nanotiter plate having a high density platen of through-hole wells with hydrophilic interiors and openings surrounded by hydrophobic material. This is described, for example, in U.S. Pat. No. 6,387,331 and U.S. Patent Application 20020094533, the contents of which are incorporated herein by reference. One specific commercial example of a nanotiter plate system is the Living Chip™ made by Biotrove, Inc. of Cambridge, Mass. Nanotiter plate technology relies on the ability to handle very small volumes of fluid samples, typically, 100 nanoliters or less. The various considerations taken into account in handling such small liquid samples are known as microfluidics.
Transferring of large collections of fluids such as libraries of small molecule drug candidates, cells, probe molecules (e.g., oligomers), and/or tissue samples stored in older style 96- or 384-well plates into more efficient high density arrays of microfluidic receptacles such as a nanotiter plate can consume one or more hours, during which time samples may evaporate, degrade or become contaminated. It is therefore advantageous to submerse the array in a bath of immiscible fluid. The fluid is ideally electrically insulating, non-conductive and nonflammable, with a relative permittivity >1. One class of fluids that serves this purpose is perfluorinated hydrocarbons, such a perfluorodecalin, perfluorooctane, perfluoropentane, longer chained perfluorocarbons or mixed populations of perfluorocarbons. Hydrocarbons or silicone fluids would also work but are flammable and tend to extract compounds from the sample.
A microfluidic volume of a liquid sample may be loaded into a target receptacle by various means. One established method for transferring a liquid sample to a surface or to another liquid uses a transfer pin loaded with the sample liquid. For example, pins or arrays of pins are typically used to spot DNA samples onto glass slides for hybridization analysis. Pins have also been used to transfer liquids such as drug candidates between microplates or onto gels (one such gel system is being developed by Discovery Partners, San Diego, Calif.). Many pin types are commercially available, of various geometries and delivery volumes. V&P Scientific of San Diego, Calif. makes slotted, grooved, cross-hatched, and other novel-geometry pins. The Stealth Pin by ArrayIt is capable of delivering hundreds of spots in succession from one sample uptake, with delivery volumes of 0.5 nL to 2.5 nL. Majer Precision Engineering sells pins having tapered tips and slots such as the MicroQuil 2000.
U.S. Pat. No. 6,149,815 describes an approach for dispensing liquid samples electrokinetically. A complex apparatus positions a receiver reservoir and a non-conducting liquid dispenser between a ground plate and a high voltage plate, neither plate being electrically connected to a sample. An accurate volume of liquid sample is transferred from the dispenser to the receiver reservoir by precisely controlling the time that a high voltage is applied to the dispenser, the longer the voltage is applied, the greater the volume of sample transferred, and vice versa. As shown in
A representative embodiment of the present invention includes a liquid dispenser for a microfluidic assay systems including systems for arraying samples for storage, screening and synthesis. The dispenser includes at least one transfer pin for transferring a microfluidic sample of liquid to a target receptacle. A pin tip at one end of the transfer pin is structured to cooperate with an opening in the target receptacle. The tip uses a high voltage potential to transfer the sample from the pin to the receptacle.
In a further embodiment, the target receptacle is one of an array of through-holes wells or closed-end wells in a platen. The target receptacle may have hydrophilic walls that attract the sample. The target receptacle may have an opening surrounded by hydrophobic material. The target receptacle may be filled with a porous hydrophilic material. A transfer pin array may include multiple transfer pins for transferring multiple samples to corresponding target receptacles. Individual transfer pins in the array may be individually actuable, as would be useful for producing patterns or layered patterns of samples. Typically the spacing of pins in the array will match a subset of a source array such as a 384 well microtiter plate as well as the spacing of the receptacle array. At least one transfer pin in the array may be independently positionable to align the at least one independently positionable pin with respect to the opening of a target receptacle. Positioning systems are typically capable of accurate movement in at least the x, y and z co-ordinates. Individual transfer pins in the array may be free floating or spring loaded.
In various embodiments, the microfluidic sample may be from 0.2 to 100 nanoliters. The transfer pin may have a diameter greater than the opening of the target receptacle. The sample may be a polar liquid such as aqueous, DMSO, dimethylformamide (DMF), or acetonitrile solutions. The high voltage potential may be between 100V and 5 kV. The at least one transfer pin may be able to dispense multiple samples without replenishment.
In a further embodiment, a voltage control module controls when the high voltage potential is applied to and removed from the pin tip. The voltage control module may operate to apply the high voltage potential to the pin tip before or after the transfer pin is positioned at the target receptacle, and to remove the high voltage potential before or after the transfer pin is moved away from the target receptacle. The voltage control module may include a resistor network and/or a controllable switch in series with the transfer pin.
Embodiments of the present invention also include a method for use in dispensing a microfluidic sample of a liquid. The method includes providing at least one transfer pin for transferring a microfluidic sample of liquid to a target receptacle. One end of the transfer pin may have a pin tip structured to cooperate with an opening in the target receptacle. Voltage is applied between the transfer pin and the target receptacle for transferring the sample from the at least one transfer pin to the target receptacle.
In such an embodiment, the target receptacle may be a through-hole well or a closed-end well in a platen array. The target receptacle also may include hydrophilic walls that attract the sample and/or an opening surrounded by hydrophobic material. The voltage may be applied to either the transfer pin or the target receptacle.
The method may also include providing a transfer pin array including multiple transfer pins for transferring multiple samples to corresponding multiple target receptacles. Individual transfer pins in the array may be individually-actuable, either sequentially or in parallel. At least one transfer pin in the array may be independently positionable for alignment with respect to the opening of a target receptacle. Individual transfer pins in the array may be free floating or spring loaded.
In such a method, the microfluidic sample may be from 0.2 to 100 nanoliters. The transfer pin may have a diameter greater than the opening of the target receptacle. The sample may be a polar liquid such as aqueous, DMSO, dimethylformamide (DMF), or acetonitrile solutions. The high voltage potential may be between 100V and 5 kV. The high voltage potential may be applied before or after the transfer pin is positioned at the target receptacle, and removed after the transfer pin is moved away from the target receptacle. The controlling step may use a resistor network and/or a controllable switch in series with the transfer pin. The at least one transfer pin may be able to dispense multiple samples without replenishment.
The method may further include applying evaporation control measures to the target receptacle. This may include immersing the target receptacle in an immiscible liquid such as a perfluorinated hydrocarbon. Alternatively, or in addition, the evaporation control measures may include at least one of humidity control, fluid pressure, and receptacle cooling.
The method may also further include positioning the transfer pin in direct contact with target receptacle, or positioning the transfer pin near the target receptacle without direct contact. The method may also include sequentially transferring multiple samples to the target receptacle to produce a layered pattern of samples.
Another embodiment of the present invention includes a microfluidic assay system. The system includes at least one liquid sample storage device including multiple storage receptacles, a microfluidic dispenser, and a dispenser positioning module. The microfluidic dispenser has a high voltage supply that develops a high voltage potential; at least one transfer pin for transferring a microfluidic sample of liquid to a target storage receptacle, one end of the transfer pin having a pin tip structured to cooperate with an opening in the target storage receptacle; and a voltage controller for applying the high voltage potential from the high voltage supply between the transfer pin and the target storage receptacle for transferring the sample from the at least one transfer pin to the target storage receptacle. The dispenser positioning module positions the liquid dispenser to enable the transfer pin to cooperate with the target receptacle for transferring the sample.
In a further such embodiment, the storage device may be a platen array of through-holes or wells. The voltage may be applied to the transfer pin or to the target storage receptacle. The target storage receptacle may include hydrophilic walls that attract the sample and/or an opening surrounded by hydrophobic material. The liquid dispenser may also include a transfer pin array including multiple transfer pins for transferring multiple samples to corresponding multiple target storage receptacles. Transfer pins in the array may be individually actuable, either sequentially or in parallel. At least one transfer pin in the array may be independently positionable for alignment with respect to the opening of a target storage receptacle. Individual transfer pins in the array also may be free floating or spring loaded.
In such a system, the microfluidic sample may be from 0.2 to 100 nanoliters. The transfer pin may have a diameter greater than the opening of the target storage receptacle. The sample may be a polar liquid such as aqueous, DMSO, dimethylformamide (DMF), or acetonitrile solutions. The high voltage potential may be between 100V and 5 kV.
The voltage controller may apply the high voltage potential to the pin tip before or after the transfer pin is positioned at the target storage receptacle, and removes the high voltage potential after the transfer pin is moved away from the target storage receptacle. The voltage controller also may use a resistor network and/or a controllable switch in series with the transfer pin.
In a system, the storage device may use evaporation control measures to control evaporation of samples from the storage receptacles. This may include immersing the storage receptacles in an immiscible liquid such as a perfluorinated hydrocarbon and/or at least one of humidity control, fluid pressure, and receptacle cooling.
The positioning module may position the dispenser so that the at least one transfer pin makes direct contact with target storage receptacle for transferring the sample, or so that the at least one transfer pin is near the target storage receptacle without direct contact for transferring the sample.
The liquid dispenser may operate to sequentially transfer multiple samples to the target storage receptacle to produce a layered pattern of samples. The at least one transfer pin may be able to dispense multiple samples without replenishment.
The present invention will be more readily understood by reference to the following detailed description taken with the accompanying drawings, in which:
Various embodiments of the present invention are directed to applying a high voltage to one or more transfer pins for transferring a microfluidic volume of a liquid sample to a suitable target receptacle. The target storage receptacle typically will have an affinity for the sample, and could be a flat surface; a surface with indentations, close ended wells, or pores; a membrane or filter; a gel; or a platen with close-ended wells or through-hole wells. In one specific embodiment, the target receptacle is one or more wells in an array of through-hole wells as part of a parallel and/or series sample transfer process. In other embodiments, the target storage receptacle may be a hydrophilic spot or divot in a hydrophobic background. Such an environment may be established on a coated glass slide such as the ones available from Erie Scientific of Portsmouth, N.H.
Typical microfluidic volumes of the through-hole wells 12 could be from 0.1 picoliter to 1 microliter, with common volumes in the range of 0.2-100 nanoliters. Capillary action or surface tension of the liquid samples may be used to load the wells 12. To enhance the drawing power of the wells 12, the target area of the receptacle, interior walls 42, may have a hydrophilic surface that attracts a liquid sample. Alternatively, the wells 12 may contain a porous hydrophilic material that attracts a liquid sample. To prevent cross-contamination (crosstalk), the exterior planar surfaces 14 of platen 10 and a layer of material 40 around the openings of wells 12 may be of a hydrophobic material. Thus, each well 12 has an interior hydrophilic region bounded at either end by a hydrophobic region.
In some systems, the well 12 may be submersed in an immiscible, non-conducting liquid such as perfluorinated hydrocarbon, hydrocarbon, or silicone fluid. An immiscible liquid prevents evaporation of samples from the wells 12 and further protects the samples from cross-communication. Of course, other evaporative control measures may also be useful, including without limitation, humidity control, fluid pressure, platen cooling, etc.
Transfer pin 20 is generally dowel-shaped, made of stainless steel, titanium, or other durable material, with a flat, rounded, tapered, or cupped tip. Typically, although not necessarily, the diameter of the transfer pin 20 is greater than the diameter of the wells 12 in order to have more rigidity in the pin and to allow the pin to reliably contact the side walls of the well. Transfer pin 20 may also have slots, grooves or spirals cut into it to increase volumetric capacity and/or to better meter the dispensing action. Transfer pin 20 may be capable of holding and/or delivering anywhere from 0.1 picoliters to more than 10 microliters, but typically holds 0.1 nanoliters to 4 microliters.
Transfer pin 20 may be free to move perpendicular to the surface 14 of the platen 10, but movement may be constrained in a plane parallel to the surface; this implementation is referred to as a floating pin. However, alternative embodiments of the invention may also be implemented with fixed transfer pins 20 as well. It is generally desirable to achieve good contact between the transfer pin 20 and the target area, but not to damage the target receptacle, well 12. This objective may be achieved by using a floating model transfer pin 20. Floating gravity-fed or spring-loaded transfer pins 20 help with reliable positioning of multiple pins to properly contact corresponding wells 12 to overcome minor errors in alignment. In some embodiments, spring-loaded transfer pins 20 may be used, preferably with “soft” springs having a spring constant that allows for relatively large displacement with a small applied force. In other embodiments, gravity-fed floating transfer pins 20 may be more advantageous in applying minimum force to a target well 12. However, gravity-fed transfer pins 20 may occasionally stick in one position following a sample dispensing cycle. One solution to this problem is to use a pressure or vacuum manifold to assist with pin positioning, such as a vacuum manifold that sucks the transfer pin 20 back into position between dispensing cycles. Floating transfer pins 20 may also use magnetism or electro-magnetism for pin positioning, such as use of a strong magnetic field for uniformly extending pins, use of magnetic pins, or by accelerating and rapidly decelerating individual pins or the entire array.
Typically, transfer pin 20 is loaded with a liquid sample for transfer to platen 10. In typical embodiments, the sample liquid may be an aqueous, DMSO, dimethylformamide (DMF), or acetonitrile solution. Then, transfer pin 20 is moved to a position over the well 12 to be loaded. The transfer pin 20 is lowered until contact is made with the opening of the well 12. When the tip of transfer pin 20 is tapered, as shown in
Once the transfer pin 20 is positioned in contact with the opening of well 12, a portion of the liquid in the pin will be wicked by capillary action into the well 12 (and displace any immiscible liquid which may previously have been stored therein). The volume of liquid sample that is transferred is self-metered by the volume of the well 12, and subject to other environmental variables, such as the action of the layers of hydrophilic and hydrophobic materials, whether the target area is under an immiscible fluid, and if so, the height of the immiscible fluid over the target area, the duration of contact with the area, the speed of withdrawal from the area, and various of the other variables listed above with respect to pin transfer.
Initializing the wicking action and wetting the interior walls 42 of the well 12 is an important point in the transfer process. Occasionally, for a variety of reasons, not all of which are well understood, there will be difficulty establishing this wicking flow. Embodiments of the present invention are directed at overcoming such difficulties in initiating the transfer of a liquid sample to a storage receptacle by applying an electric potential. Although this approach may be useful for non-polar liquids, it is especially useful for transferring samples of polar liquids such as aqueous, DMSO, dimethylformamide (DMF), or acetonitrile solutions can be transferred into a target well 12 by contacting a transfer pin 20 filled with sample and applying a high voltage with low current (typically less than 5 microamps).
Embodiments of the present invention use the existing transfer pin and platen well arrangement described with respect to
The voltage necessary to effect sample transfer depends on the physical properties of the sample and the receptacle, i.e., well 12, including their affinity for each other. In addition, the choice between AC and DC voltage supplies may affect the voltage necessary for transfer of a liquid sample, but both types of supplies are acceptable. Generally, the voltage will be between 10V and 50 kV, typically in the range of 100V to 5 kV. The choice of voltage level is affected by effects of ohmic-related heating and material breakdown characteristics. With a high dielectric constant liquid, a high voltage of large voltage pulse may be applied without electrical breakdown.
It is desirable to limit the current flowing from the transfer pin 20 in order to prevent electrical heating, etching and ionization of the sample, receptacle, pins, air, or immiscible fluid. Therefore, it is important to use a high-voltage, low current system. Examples high-voltage, low current sources include a Van De Graaf generator, or a standard high voltage source in series with a high-voltage, high-resistance resistor.
In one specific embodiment, the voltage is applied to the transfer pin 20 after it is positioned at the opening of the desired well 12, and the voltage is removed after the sample has been transferred to the well 12 and the transfer pin 20 has been withdrawn from the opening of the well 12. In other embodiments, the voltage may be applied to the transfer pin 20 before it is positioned at the opening of the desired well 12, and the voltage is removed after the sample has been transferred to the well 12 but before the transfer pin 20 has been withdrawn from the opening of the well 12.
In addition, voltage aided sample transfer in various embodiments may be based on either full, partial, or no physical contact between the transfer pin 20 and the target well 12. That is, in some embodiments, the end of the transfer pin 20 may be brought into substantial physical contact with a portion of the target well 12 in order to transfer a liquid sample from the pin to the well. In other embodiments, the transfer pin 20 approaches the opening of the target well 12 without actually establishing significant contact in order to transfer a liquid sample from the pin to the well. Some embodiments with or without contact may benefit from electrospray effect to transfer a sample from the transfer pin 20 to the target well 12.
In various embodiments, either the target well 12 or the entire platen 10 may be electrically grounded. In other embodiments, the platen 10 and well 12 may be ungrounded. Either approach may be successful so long as there is an appropriate voltage difference between the transfer pin 20 and the target well 12. In addition, the platen 10, itself, may be made of conductive material, or non-conductive material. Moreover, specific embodiments may not necessarily require a combination of hydrophilic and hydrophobic materials as described with respect to
The efficiency of voltage aided sample transfer also may depend on the relative geometries of the transfer pin 20 and the target well 12. For example, a transfer pin 20 with a tapered point such as shown in
In addition to use of an individual transfer pin 20 as shown in
The top side of each of the transfer pins 20 may be electrically connected either directly or via a resistor, switch, or transistor to a voltage source. The voltage may be specific for each transfer pin 20, or multiple transfer pins 20 may share a common voltage source.
The top sides of the transfer pins 20 are electrically connected to pin voltage sources 36 in a voltage control array 34, which may optionally include a voltage control port 38 addressable by an external processor. Each individual pin voltage source 36 may be, for example, a resistor element in a resistor network (i.e., the voltage control array 34) connected to a high voltage source so that each transfer pin 20 is connected via its own resistor to the high voltage source. To reduce the cost and size of the system, a single source resistor may be placed between the high voltage source and the resistor network, which allows the use of smaller, cheaper lower resistance resistors in the network together with a single bulky, more expensive, high-resistance resistor at the source. For example, the source resistor could be a 1 to 10 gigohm resistor, and the pin resistors could be 1 to 10 megohms each. However, it may be advantageous in terms of uniformity of transfer throughout the pin array 30 to have a higher resistance on the pin resistors, for example each pin having a gigohm resistor.
To individually actuate at least one transfer pin 20 using voltage application, a controllable switch may be placed in series with each actuable pin. These switches may be, for example, high voltage transistors or relays, and also may be controlled by a microprocessor. In one specific embodiment, each spring-loaded transfer pin 20 may be loaded on a spring, which also acts as an electrical contact to a printed circuit board voltage control array 34. The printed circuit board voltage control array 34 may contain the resistor network and connections to the high voltage source. In some embodiments, the printed circuit board voltage control array 34 also may contain the switch networks and connections to the computer or other device for selecting a sample dispensing pattern.
Thus, in one embodiment, each transfer pin 20 in a multiple pin array 30 is individually addressable for purposes of applying a high voltage potential to the pin. In such a pin array 30, one transfer pin 20 at a time may be actuable, multiple pins may be actuable at one time, or all of the pins in the array may be actuable at one time. The more transfer pins 20 that are actuated at any one time, the greater the parallel processing of the system. By actuating different patterns of multiple transfer pins 20 (in a manner analogous to an ink jet computer printer) patterns of samples may be developed. By repeating this process, layered patterns may be developed, including the synthesis of organic molecules such as peptides, small molecules or oligonucleotides.
In another embodiment, a pin array may be equipped with a controller for selectively extending or retracting a subset of transfer pins 20 to cause contact or removal from contact of those pins for the purpose of dispensing a pattern of sample. For example, an array of solenoids could be used to retract those transfer pins 20 that are not desired to contact the receptacle well 12. The solenoids may act directly on the transfer pin 20, or by a remote drive mechanism such as an array of pistons positioned slidably in an array of tubes. Alternatively, an array of controllable valves connected to a vacuum or pressure manifold may be used to selectively retract or extend a subset of transfer pins 20. Moving the pins in the array 30 so that only transfer pins 20 selected for sample transfer approach the opening of selected wells 12 avoids inadvertent transfer of a liquid samples from non-selected pins to non-selected wells, such as by wetting, which may occur even when no voltage is applied to a non-selected pin. It may be desirable to both selectively actuate a pattern of transfer pins 20 using both movement controllers and application of high voltage to the selected pins in order to prevent inadvertent dispensing, such as by electrospray.
Although various exemplary embodiments of the invention have been disclosed, it should be apparent to those skilled in the art that various changes and modifications can be made which will achieve some of the advantages of the invention without departing from the true scope of the invention.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10227179 | Aug 2002 | US |
| Child | 12148416 | US |
