Acoustic dispensing expands the microarray toolbox. Drop-on-drop delivery is difficult to accomplish with pins without contamination but is readily handled by acoustic dispensing for the stepwise addition of reagents. We demonstrated accuracy and precision by dispensing AMC and FITC solutions into glycerol nanodroplets. The energies used for each individual sample were optimized to produce good spot morphology and centering, two factors that are critical to the integrity of the microarray and for targeting subsequent reagents into existing spots. The volume of each droplet was determined by dispensing fluorescent dyes of known concentration into a known well volume and comparing to a calibration curve. Real-time scrutiny through the camera showed that the dyes were dispensed directly into the center of each glycerol spot, and CVs were < 5%; a comparable approach with pins produced CVs > 16%18
. This capability encouraged a push toward the assembly of more complex, enzymatic bioassays; we started with cathepsin L protease as it and its associated inhibitor were both well-studied in our laboratory. The resulting dose-response behavior from this proof-of-concept experiment was indicative that the reactions were properly assembled, paralleling those constructed by liquid handlers in well plates or by hand in test tubes.
We can transfer 1-nL volumes of aqueous-, DMSO-, and glycerol-based working buffers while maintaining the proper morphology and spatial-targeting control required for microarraying. A single 2-nL glycerol spot has a diameter of approximately 200 μm, and variable sizes can be achieved by increasing the number of dispenses to a particular location. The droplets land on one another and coalesce to form a single spot. Larger reaction volume compartments are easier to target when introducing subsequent reagents for multicomponent assembly. Moreover, the increased amount of sample in each spot ultimately results in a stronger signal. Pins are not capable of variable volume transfers, and pin arrayers generally produce microarrays in a regular, rectangular pattern, especially if multiple pins are used. Acoustic dispensers allow for complex arrangements as sample from any source well can be transferred to any location on the target surface. The ability to spatially dispense to a chip from 384 or 1536 wells is an application that is useful for cherry picking a compound plate to screen an enzyme assay or perhaps to dispense samples onto specific areas of a tissue slice, for example.
When aqueous or organic buffers are printed by a pin, often each successive spot displays a decrease in diameter as the pin empties. And for glycerol-containing samples, spots that are printed immediately following a reload often have larger diameters than those printed prior to the reload, unless an extensive preprint or blotting is performed, which results in wasted sample and extended run times27
. These factors contribute to greater CVs. Acoustic dispensing does not suffer from this effect as each spot is ejected fresh. The geometries of acoustically dispensed arrays are not quite as symmetric as those generated by pins as frequently the spot moves in a random direction after it settles on the substrate surface; aqueous components evaporating out of the glycerol-containing spots and non-uniformities on the solid surface contribute to this shifting. However, provided the center-to-center spacing is sufficient, occasional shifting rarely causes two spots to merge, and most genomics and microarray software packages can easily account for these minor misalignments during analysis. The step motor on a pin arrayer can have 5 - 10 μm precision in the x- and y-directions so the spots are usually spaced out very accurately.
The pin’s size, material, and chemical coating should be chosen carefully to achieve the optimal physical interaction with the sample and substrate during a print run. Other factors that affect spot size include the depth and time the pin dips into the sample, number of preprint blots, and the contact time and retraction speed on the substrate surface37
. Additionally, the volume transferred and spot morphology may depend on the chemical coating of the solid surface for contact methods, and better spot morphologies are evident when no-contact methods print on hydrophobic surfaces19
. There is also a very little chance of cross-contamination or carryover with no-contact microarraying as is possible when a split pin dips into multiple wells during a long print run. To combat this, microarrayers have several built-in units for cleaning the pins, including sonicators, wash stations, and dryers. Since several loops are usually required, the washing, drying, reloading, and preprinting steps may contribute greatly to the total run time when a single pin is employed18
Acoustic dispensing eases the use of certain solutions where contact may not be desired as the reagent would damage the pin or contaminate it with something that is difficult to remove; very acidic or basic solutions, “sticky” biological proteins or beads, and radio-labeled compounds fall under this category (contact is made with the sample by the microarraying technologies mentioned earlier, which all have tips, nozzles, or tubing). Furthermore, a pin interacting with a protein may have an effect on its kinetic properties and folding state, and may cause denaturation30,38
. Complex biological samples containing proteins or cells may experience electrostatic interactions or adhesion forces that lead to a higher localized concentration on the surface of the pin, preventing transfer to the slide, and resulting in a lack of homogeneity between spots on the microarray26,30
. These concerns are minimized with acoustic dispensing, and we demonstrated with a non-homogeneous suspension of fluorescent, 1-μm diameter beads that it can deliver spots that are representative of the sample in the source well. Spanning a range from 0.1 to 10 ×108
beads/mL, there was a direct, linear relationship between concentration in source well and number of beads transferred in each spot.
Viscous solutions containing upward of 25% glycerol by volume can be handled before the acoustic dispensing capabilities start to break down. Our reaction buffers typically contain 10% glycerol by volume (to prevent evaporation of the spots), and at this level there are no problems with the dispensing. When we transferred cellular lysate solutions, the presence of detergents led to difficulties in producing spots that did not fragment (this would be less of a problem dispensing into well plates as morphology would not be a concern). To overcome the formation of satellites, the concentration of detergent should be reduced and some glycerol may be added to try and improve performance. Spotting at a high glycerol concentration is not a problem for pins, and we have regularly used 50% glycerol solutions for protease assays18,23
. It may be difficult to transfer lysates to the substrate surface due to the complexity of the sample and possible physical interactions with the pin, and thus clever design is required for lysate microarrays36
Acoustic dispensing requires flat-bottom polypropylene or COC plates, both of which are ideal for compound storage. They can be 384-, 1536-, or 3456-well formats, and for 1536-well plates, only 0.25 mm of liquid sample is needed for dispensing to be possible (the tool can detect the amount of sample remaining in the well), corresponding to a volume of about 1 μL. Even after sampling a plate multiple times, the leftover liquid can be stored for future use until the wells are drained, minimizing wasted material. This reduces the cost for microarraying expensive or rare proteins, and allows for the repeated use of compound plates. In sharp contrast, contact arrayers require volumes of 5 - 10 μL and 25 - 50 μL for 384- and 96-well plates respectively.
Three slides can be placed into the acoustic dispenser at one time and array densities of approximately 1000 - 3000 spots/slide can be achieved. This platform cannot match the throughput or density of pins and piezoelectric dispensers. As a microarrying tool, acoustic dispensing is best utilized as a low-throughput platform for specialized applications that require complex assembly, use expensive reagents, or function better with minimal contact with solid surfaces. The technology was an upgrade over an existing screening platform developed in our laboratory involving the aerosol deposition of proteases to activate microarray reactions because with acoustic dispensing we are able to determine the exact volume of enzyme introduced to each spot, and only a fraction of the sample is required18,23
. We have also shown that kinase bioassays can be constructed and analyzed39