This invention relates generally to an apparatus and method for fabricating microarrays of biological samples on a support substrate, and more particularly to a dual manifold system for the rapid, parallel transfer of reagents to test substrates for large-scale screening assays.
In clinical chemistry, it is frequently necessary to carry out the metered application of an analytical liquid to a target. One case which is particularly relevant to the present invention is the application of the analytical liquid to an analysis element such as a chip-based biological sensor in which biological materials are integrated with microelectronic devices. In recent years, rapid technological advances have enabled the use of micro-scale chemical/biochemical reactions for performing various types of analyses. For instance, DNA microarrays such as genosensors allow thousands of samples to be assessed simultaneously on a microelectronic test chip that is less than one-quarter of an inch in length per side. Typical test sites on such a chip are on the order of about 100 microns (xcexcm) in diameter. Conventional applications of chip-based biological sensors include mutation diagnosis, organism identification and gene expression profiling. More recent applications, such as parallel screening of chemical compounds for drug discovery and protein arrays for functional analysis, will soon be routine.
Known fluid handling systems for dispensing, or xe2x80x9cmicro-spottingxe2x80x9d, arrays of biological materials on a target substrate commonly comprise pick-and-place equipment. Generally, pick-and-place dispense systems include a dispense head adapted for transferring volumes of fluid from a fluid source to a target substrate. The time required to pick up, transfer and deposit a given volume of liquid significantly limits the efficiency of pick-and-place systems for micro-spotting. This lack of efficiency is even more pronounced where the target substrate contains hundreds, or even thousands, of test sites. Efforts have been made to improve the efficiency of pick and place systems for micro-spotting. For instance, systems have been adapted for picking up, transferring and depositing multiple sample volumes simultaneously. However, the time required for dispense head movement remains a significant limitation of such systems.
Furthermore, the multiple degrees of freedom associated with the movement of individual system components, such as the dispense head, significantly limits the positional accuracy of samples deposited on a target substrate. In instances where the equipment is adapted for contact dispensing (i.e., where the dispense elements of the system physically contact the target substrate to effect transfer of the fluid to the target substrate) such limitations may be magnified. In particular, dispense tip deformation can lead to irregular sample spacing and, in some instances, cross-contamination of adjacent test sites.
Due in part to the aforementioned limitations, the positional accuracy and liquid transfer volume capability of conventional pick-and-place dispensing systems can not meet the requirements of many evolving applications. Constructing microarrays having a higher degree of miniaturization will require an increase in test site array density. Realizing such an increase in density will require a reduction in sample spot size and spot pitch (i.e., the center-to-center distance between adjacent deposits). In order to achieve such miniaturization, a fluid handling system capable of accurately and efficiently depositing chemical and biochemical reagent droplets having volumes on the order of picoliters is required.
Technology for dispensing liquid volumes on the order of picoliters exists, but has been primarily limited to the field of ink-jet printing. Many drop-on-demand ink-jet ideas and systems were invented, developed, and produced commercially in the 1970s and 1980s. A detailed and comprehensive summary of state-of-the-art drop-on-demand ink-jet printing technologies, including the fabrication of ink-jet valves and printheads, is provided in a published article by Hue P. Le, entitled Progress and Trends in Ink-jet Printing (Journal of Imaging Science and Technology, Volume 42, Number 1, pp. 49-62)(1998).
There is an established need for an apparatus and method for accurately and efficiently transferring and depositing, or printing, microarrays of reagent samples having volumes on the order of picoliters on a test substrate. It would be desirable to have a microarray printing apparatus for performing large-scale chemical/biochemical screening assays, wherein the system incorporates known drop-on-demand ink-jet printing technology and is particularly suited for dispensing chemical and/or biochemical reagents.
It is an object of this invention to provide a liquid transfer apparatus capable of accurately and efficiently transferring liquid reagents from an array of reservoirs to an array of sites on a target substrate
It is another object of this invention to provide a liquid transfer apparatus capable of accurately and efficiently depositing volumes of liquid reagents in the range of about (10)xe2x88x9212 to about (10)xe2x88x926 liters.
It is another object of this invention to provide a liquid transfer apparatus capable of effecting such reagent transfer with minimal movement of the apparatus during operation.
It is another object of this invention to provide a liquid transfer apparatus employing non-contact dispensing.
It is another object of this invention to provide a liquid transfer apparatus and method adapted for the automated printing, or micro-spotting, of multiple analytical chips in succession for performing large-scale screening assays.
These and other objects are achieved with the assembly and method of the present invention. Briefly, according to the invention, a dual-manifold assembly generally includes an aspiration manifold 10, a dispense manifold 20, and fluid transfer elements 80 for the parallel transfer of fluids therebetween. Although the apparatus and method are adaptable for use transferring a variety of liquids to a variety of target substrates, in the preferred embodiment of the invention the apparatus is particularly suited for transferring chemical or biochemical reagents from an array of microtiter plate wells to an array of test sites on a chip-based biological sensor.
The aspiration manifold 10 is positioned above a source plate 50, such as a microtiter plate, and is adapted for simultaneously aspirating liquid, such as a chemical reagent, from an array of reservoirs 52. In the preferred embodiment of the present invention, the aspiration manifold includes an array of aspiration manifold subassemblies extending through a base plate 17 and adapted for being received by an array of reagent-filled wells 52. When the aspiration manifold is seated onto the microtiter plate 50, each subassembly seals a single well such that fluid communication to and from the well is limited to a pair of conduits 12, 14 extending into the well. In operation, each well is pressurized by a pressure source 40 through conduit 12 which urges the liquid 54 through conduit 14 toward dispense manifold 20. In an alternate embodiment of the invention, the aspiration manifold has a gasket element 23 for sealing against the periphery of the microtiter plate 50 during operation, precluding the need to pressurize the wells individually. In this alternate embodiment, pressurization of the wells 52 is accomplished through a single pressure conduit 12 extending through base plate 17.
In the preferred embodiment of the invention, a plurality of aspiration conduits communicate with the dispense manifold side of the assembly through a modular connector 90. Generally, the modular connector includes a male component 94 which releasably engages a female component 92. Preferably, the aspiration conduits 14 terminate at component 94 which has integral tips 95 adapted for receipt by female component 92. Preferably, the female component 92 of the modular connector is integrated into valve assembly 21. Conduits 24, 26 and 38, of valve assembly 21, can also be adapted for modular connection with other subassemblies of the dual manifold system. For instance, although dispense manifold 20 can be directly integrated into valve assembly 21, in the preferred embodiment of the invention dispense manifold 20 is adapted for modular connection to valve assembly 21.
Volumes of reagent in the range of about (10)xe2x88x9212 to about (10)xe2x88x926 liters are ejected from the dispense manifold through an array of dispense manifold orifices 106 upon application of a force, such as a pressure pulse. Preferably, a pressure pulse is provided by a valve 28 incorporating ink jet style drop-on-demand technology. Valve 28 may be adapted for modular connection to valve assembly 21 or, alternatively, valve 28 may be directly integrated into the valve assembly. In the preferred embodiment of the invention, the dispense manifold 20 comprises a standard ink-jet style printing head having micro-machined channels each terminating at an orifice 106.
In an alternate embodiment of the invention, a purging apparatus 30 is provided for periodically purging the assembly. Purging apparatus 30 includes gas and cleaning liquid inlets, 32 and 34 respectively, controllable through a solenoid or other suitable valve 36. Preferably, purging apparatus 30 is adapted for modular connection to valve assembly 21. Alternatively, apparatus 30 can be directly integrated into the valve assembly.
In further embodiments of the invention, auxiliary features are included for improving registration and alignment of reagent samples deposited on the test chip. In particular, fiducial pins and/or marks are integrated into the target substrate. The integrated features are sensed by a conventional vision system for ensuring proper alignment and orientation during operation.
In yet a further embodiment of the invention, detection means are provided for detecting the passage of a droplet 104 of reagent from a dispense orifice 106. The detection means may comprise optoelectronic devices, such as a photodiode 100/photodetector 102 pair positioned near each orifice. Alternatively, electronic conduction-based sensors can be integrated into the dispense manifold.
In operation, microtiter plate 50 is positioned below the aspiration manifold 10 and the aspiration manifold is subsequently seated onto the plate such that an air-tight seal is formed between the aspiration manifold and the plate. Microtiter plate wells 52 are pressurized by a pressure source 40 through at least one conduit 12 to effect the transfer of reagent through a plurality of conduits 14 and into dispense manifold 20 positioned above target substrate 60. Subsequently, a pressure pulse from valve 28 is communicated to the dispense manifold 20, effecting the transfer of a desired volume of reagent through orifice 106 to test sites 64 on target substrate 60. Further embodiments of the method include aligning the target substrate to ensure proper positioning of the reagent deposits, detecting the passage of reagent samples from the orifices of the dispense manifold, and purging the system between deposition, or printing, operations.