Advantages of Ink-Jet Dispensing. The ability to dispense 25-100µm droplets using ink-jet technology equates to a single droplet volume resolution of 10pl to 0.5nl. [Note that this range represents the design space of dispensing systems, not the operating space of a specific system.] Reducing volumes from the 1µl range to the 0.1nl range would reduce consumption and waste of rare and expensive materials by 10,000-fold. Moreover, reducing the size of chemical test reaction sites to small, printed microspots would permit the creation of massively-parallel, high-density arrays of multiple test sites. Such arrays will allow fast, automated test outcome detection. This would increase throughput drastically, again lowering cost.
For these biochemical reaction arrays to be low cost, a singe machine would have to be able to produce large numbers of arrays at a time. The maximum droplet creation rates achievable with demand mode ink-jet dispensing, 8kHz or more, are probably higher than could be utilized in an array printing system because of the high stage speeds required: fluid droplets dispensed on 200µm centers at 5kHz translates to a 10m/s stage speed. However, even at 100Hz, a hundred-element array could be produced roughly every second.
As a non-contact printing process, the accuracy of ink-jet dispensing is not affected by how the fluid wets a substrate as is the case when positive displacement or pin transfer systems "touching off" the fluid onto the substrate during the dispensing event. In addition, fluid source cannot be contaminated by substrate, or contamination on the substrate, in a non-contact dispensing process. Finally, the ability to free-fly the droplets of fluid over several millimeter allows fluids to be dispensed into wells or other substrate features (e.g., features that are created to control wetting and spreading).
Potential and Problems in Using Ink-Jet Dispensing in Drug Discovery.
The recent emphasis on combinatorial methods and high throughput screening for drug discovery has created a strong motivation to substantially reduce the fluid volumes used, both to lower cost and to increase throughput. Currently, 96 and 384 well microtitre plates represent the state of the art in automated, miniaturized, high-throughput biochemical systems. Ink-jet technology can fulfill reduce volumes to the point where material cost is not a significant factor. To illustrate the degree to which ink-jet microdispensing could further reduce the size/volume scales, Figure 6 shows ink printed on the bottom of a 96 well plate in the form of characters. The 60µm drops used produce 100µm spots and individual spots are visible in the blow-up of the T at lower right of the image.
Figure 6
Reactive Materials
- Liquid metals
- Fluxes, photoresists
- Poxies, UV cured materials
Organic Solvents
- Alcohol
- Keytones
- Aliphatics
- Aromatics
- Dipolar solvents
- Lipids
Biological Fluids
- DNA solutions
- Antibody solutions
- Buffers, solvents, etc.
Other
- Sol-gels
- Thermoplastics
- Particle laden fluids
- Latex beads
- Metal particles
Achieving the ultimate potential of ink-jet microdispensing will depend on resolving a number of issues, material compatibility being one of the most important. This is especially the case for chemical synthesis, where a wide range of materials is used. Each ink-jet printhead/dispensing system would have to be designed specifically for the materials that it is expected to dispense. To illustrate range of materials that can be dispensed, Table 1 shows the types of materials that MicroFab has successfully dispensed. Some of these materials are components of both water-based and solvent-based inks used in our array printhead development efforts. The others are a result of our efforts to apply ink-jet dispensing to electronics manufacturing processes (12), micro-optical element fabrication (13), laser surgical procedures (14), and medical diagnostics manufacturing.
Examples of the printing of these materials listed in Table 1 are shown in the adjacent figures. Figure 7 shows features etched in a polymer by dispensing acetone using ink-jet technology. Figure 8 shows solder bumps that were created by ink-jet dispensing of molten solder onto the silicon substrate. Figure 9 shows an array of thermoplastic microlenses printed with ink-jet technology.
Figure
7
Figure
8
Figure
9
DNA Probe Array Fabrication: An illustrative Example.
As part of a National Institute of Standards and Technologies Advanced Technology Program grant to the Genosensor Consortium, MicroFab has developed an ink-jet based oligonucleotide array printer (15). Eight modular dispensing units, each with an integral 500µl reservoir, can be installed on the printer at a time. Each of the eight dispensers can be addressed by the drive and control electronics via a PC, but only one of the eight can be active at a time. The modular dispensing modules are mounted on a plate so that groups of eight dispensers can be changed easily while maintaining their alignment. Figure 10 illustrates the system and Figure 11 shows the modular dispensing units.
Figure
10
Figure
11 To date, we have successfully printed oligonucleotide probe arrays containing thirteen different probes and have made arrays using sixteen different dispensing devices (all using the same fluid, a fluorescent ink). In addition, we have fabricated arrays of dye spots of different sizes and spacings to support the imaging development efforts. Figure 12 illustrates the results of a proof-of-principle experiment where four different synthetic 9-mer oligonucleotide probes have been attached in 100µm spots on 500µm centers, hybridized to a known, S35 labeled synthetic 9-mer target, and imaged on a CCD detector by placing the sample directly on top of the CCD array (i.e., in proximal contact mode; image courtesy of MIT Lincoln Laboratories). The first column is a G & A mismatch at the end of the 9-mer, the second is a perfect match, the third a C mismatch, fourth an A mismatch. The pattern is then repeated.
Figure
12
In another collaborative project, MicroFab will participate in feasibility studies to design DNA diagnostic probe arrays for identifying infectious pathogens (in collaboration with D.D. Dao of the Houston Advanced Research Center and J.M. Musser of the Baylor College of Medicine). This project involves printing hundreds of allele-specific oligonu cleotides onto a 1cm2 test substrate for the purpose of identifying the species and strain of multiple drug resistant Mycobacterium tuberculosis infections.
High Density Dispensing Array
for High Throughput Applications
Although the system and dispensing devices shown in Figure 10 and Figure 11 could conceivably be used in applications requiring the dispensing of hundreds of different types of fluids, the increase in complexity due to the large number of devices involved makes this approach questionable. For applications requiring a large number of fluids, an integrated multiple fluid dispenser is required.
MicroFab has developed array drop-on-demand piezoelectric ink-jet print head technology for use in high speed, photorealistic printing. Methods have been developed to fabricate 120 individual dispensing channels into a line array of less than one inch (170µm spacings). Channel-to-channel droplet velocity uniformity (and by inference, droplet volume) of better than 5% has been demonstrated. Printheads with sixteen channels for each of four different fluid (CMYK inks) have been built into line arrays of less than one inch and used to create high quality color images. The technology embodied by this print head is described in 17 U.S. patents, with 25 pending (16).
The printhead construction is described briefly as follows: first, two small blocks of piezoelectric material are laminated together with electrically conductive epoxy such that their poling directions are aligned. This "sandwich" structure is then machined with a precision diamond saw, creating both fluid channel openings and channel actuator structures. Both the channel and wall widths for the array are 85µm, and channel depth/wall height are 400µm.
After sawing, the tops of the ink channels are formed by attaching a cover plate to the machined sandwich structure. The cover plate is solid except for the regions that are machined to form the fluid manifold (manifolds in the multiple fluid configuration).
An excimer laser ablation process is used to machine precision (±0.5µm) 40µm diameter orifices into an adhesive backed polymer film that attaches to one end of the printhead. The orifices are machined to align to the fluid channels when assembled. The printhead is fluidically complete when the back of the channels are sealed with an acoustic energy absorbing polymer. Figure 13 illustrates these process steps, and an example of an actual parts is shown in Figure 14
Figure
13
Figure
14The final fabrication step is electrical interconnect. When the sandwich structure is formed, the lower piezoelectric block is longer at the rear (i.e., the surface opposite from the orifice plate) of the printhead, thus leaving a metallization layer exposed. The sawing process thus creates individual metallized bonding pads. A flex-circuit will be attached to these pads, allowing the metallization layer in the middle of each channel wall (i.e., actuator) to be individually addressed electrically. Figure 15 illustrates the printhead in operation.
Figure
15As part of this print head technology developments effort, we have developed technology for dynamically modulating the drop volume. A 4:1 range of volume modulation has been achieved to date, resulting in a 4:1 range in printed spot area. The modulation is continuous (i.e., not discrete) over a significant part of the total range, and is achieved at printing rates up to 3,000 drops per second (17). In biomedical applications, this new technology will permit solution volume and concentration to be varied over a continuous range on individual microspots, giving precise control of reaction conditions for studies of binding kinetics or affinity.
MicroFab is currently formulating a project to adapt this high density, multichannel print head technology to DNA and other diagnostic array fabrication applications. The initial configuration will have sixteen distinct fluid inputs, each feeding a single dispensing channel in a line array less than one inch long. Developments in this area will eventually produce a multiple fluid microdispensing capability that will have extremely high throughput capabilities and real time volume modulation capabilities.
Drug screening tests using multichannel printheads could be extremely rapid. With reagent dead volumes of only a few µl, and a capacity of 100's of separately-dispensed fluids in a few (single?) dispenser heads, a multichannel bioprinter could dispense all of the concentration X drug samples for a 10-level receptor-binding assay for hundreds of different candidate drugs in just a few seconds. Combined with optical detection of binding results (14), such a system could screen large numbers of candidate molecules for biological activity with unprecedented speed and efficiency.