Abstract
Acoustic droplet ejection (ADE) gently and precisely aliquots nanoliter and picoliter liquid volumes without any physical contact with the solution being transferred. The technology is very automation-friendly, as it is compatible with conventional microplates. Focused energy from an acoustic transducer induces droplet ejection into an inverted standard microplate. The commercial system transfers low-nanoliter volumes of dimethyl sulfoxide–dissolved compound libraries and thereby enables cell-based assays to be performed in 1536-well plates.
Introduction
Robotic systems and standardization of fluid containers have facilitated the industrialization of genomics, proteomics, and high-throughput screening (HTS). Automation of life science research and development has reduced the assay volume from the milliliter to the microliter scale, but further reduction, although highly desirable, is largely stalled by the lack of reliable systems for transferring nanoliter and picoliter volumes. In addition to lacking reproducibility in the low-nanoliter range, currently available systems are not compatible with many life science applications because of their destructive impact on living cells and fragile macromolecules. With the increase in cell-based assays, gentle transfer techniques are of increasing interest. Also, due to the impact of dimethyl sulfoxide (DMSO) on cell assays, reducing the volume of DMSO required to transfer a compound of interest into an assay is desired, and this is often a barrier to reducing total assay volume.
The use of focused acoustic energy to transfer liquids in the nanoliter and picoliter volumes overcomes these issues, as it is precise, reliable, and gentle enough to move life science materials, including cells. The process scales down to the low nanoliters, enabling cell-based assays in 1536-well volumes with 0.1% DMSO concentration. Acoustic energy reflected from the well and fluid interfaces can be used to audit well volume and solvent composition, providing information for both process and quality control.
This article discusses the acoustic drop ejection technology and explores the unique aspects of focused acoustic energy transfer of droplets from source microwells upward into an inverted receiver well. In particular, automated plate inversion, holding capacities of inverted plates, and transfer into dry wells are explored.
History of Acoustics for Liquid Handling
The origin of liquid transfer with acoustic energy dates back to early experiments with high-intensity acoustic beams at the Tuxedo Park laboratory of Alfred Lee Loomis in 1927 where it was observed that immersing a high-power acoustic generator in an oil bath would create a mound at the surface “erupting oil droplets like a miniature volcano.” 1 (An interesting historical account of the research at this famous lab was recently published. 2 ) Improvements in the 1950s and early 1960s localized the energy with “exponential” acoustic horns or focused it with acoustic lenses but still maintained the high intensity of earlier devices. These devices create drops with continuous application of acoustic energy to form geysers of small droplets or patterns of disturbances on the liquid surface where some of the swells grow large enough to pinch off and become drops. 3,4 This process was used in commercial nebulizers to make mists of medications for inhalation in the late 1960s. 5,6
The introduction of a lower-intensity process that both focused and pulsed the acoustic energy in order to create a single “drop-on-demand” was developed in the early 1970s. 7 Here a single acoustic lens was used in direct contact with ink in order to create a droplet when an ink pixel was desired on a printed page. The technology has been extended to multiple lenses and multiple inks for printing since the late 1970s. 8,9
Acoustics for Life Science Liquid Handling
The crossover of advances in focused-acoustic, drop-on-demand technology back to biological materials arose as life science research became increasingly automated during the 1990s to support the need for precise and reliable, robotic liquid dispensing. Efforts to adapt acoustic drop ejection technology (ADE) for “industrialized” life science liquid handling based on the microplate began in earnest in the 2000s. 10
An initial application of ADE in life sciences is transferring nanoliter or picoliter droplets from conventional flat-bottom microplates using a focused acoustic transducer (see Figure 1). This is accomplished without placing the transducer in the microplate. Rather, the transducer is located below the microplate where it can move freely from well to well. When positioned under the desired well, the transducer receives radio frequency energy in the megahertz range that is converted into an acoustic vibration. If the transducer height is adjusted to bring the focus just below the surface of the liquid in a well of the microplate, the acoustic vibration ejects a droplet of precise volume. Ejected droplets can then be captured by a receiving microplate or any other surface positioned in the path of the droplet.
The acoustic droplet ejection transfers nanoliter or picoliter droplets from a conventional flat-bottom microplate (A) using acoustic energy (B) from the transducer (C). The energy is focused on the surface of the liquid in the lower well, causing a droplet of precise volume to be ejected without any physical contact between the transducer and the liquid being transferred. Drops can then be captured by empty “dry” wells or filled “wet” wells.
Along its path to the liquid surface in the microplate well, the acoustic waves propagate through multiple materials. First, they enter a conducting rod containing an acoustic lens. The lens focuses the waves into a coupling fluid that is typically water; the coupling fluid is present to provide a more efficient path than air for the acoustic energy to bridge the gap from the lens to the microplate bottom. The coupling fluid slowly flows over the lens face and then falls into a catch tank below. Any excess coupling water is removed from the microplate bottom by a vacuum wiping system. After traversing the coupling fluid and the microplate bottom, the acoustic waves enter the well fluid and head towards a focal point near the fluid surface to eject a droplet.
Ejection Process
A stroboscopic capture image of the drop ejection process is shown in Figure 2. In each of the four panels shown, a strobe is flashed multiple times during the flight of a single 5 nL droplet of DMSO as it travels from the source fluid well to a receiving surface (a glass slide in this case to improve visibility) at 1 m/s. The first drop is shown attached to the slide in the upper left hand corner of the figure, and the second drop was captured by the strobe as it travels to meet it. The second drop and all subsequent drops coalesce into the deposited liquid without generating any secondary droplets or splatter.
Acoustic ejection of a 5-nL droplet (212 μm diameter) of DMSO traveling upwards at approximately I m/s to a glass slide with either 1, 20, 40, or 100 drops already deposited. Each droplet is captured at six points in time using a multi-flash strobe at intervals of 400 μs. The critical impact Weber numbers for the droplets are between 4 and 5 and, as expected, no evidence of splatter is found for either a dry deposition on left or for receiving into the 0.5 μL drop on right.
The shape of the source well meniscus relaxes after drop formation, and the consistent nature of the ejection process is illustrated at the bottom of the figure by the superposed meniscus states of the multistrobe images. Drop volume reflects this reproducibility and typically has coefficients of variation (CV) below 2%. A more detailed discussion of volume transfer precision for compound library reformatting is given below.
Wet or Dry Wells
For HTS, the acoustically generated drops are captured in receiving microplates, and like the glass slide of Figure 2, receiving microplate wells may be “dry” or “wet”—that is they can either be empty or prefilled with assay components. Dry plate transfer is often desirable when plates will be shipped or stored for later use. Transfers into wet wells are quite common, yet transfers into inverted wet wells are less familiar. Inverted transfers raise concerns over splashing as a source of contamination since any droplets formed from a splash will not be pulled back into the well by gravity.
Empirical studies show the DMSO drops from ADE merge into the inverted well fluid without creation of detached droplets (there are no recoil drops or crowning). Drop coalescence in acoustic transfers is also consistent with fluid-dynamical theory as shown in the “regime” map of Figure 3 due to the relative dominance of surface tension forces over the droplet inertial forces. In particular, the dimensionless parameter of Weber number defined as We = %rHU2D/σ (where p is the drop liquid density, U is the velocity, D is the drop diameter, and σ is the surface tension) provides a relative measure for surface tension and inertial influences on the drop dynamics, and the “critical impact Weber number,” or Wec, is the value above which splashing occurs.
Surface tension will hold liquids and retain droplets in inverted wells. This “regime” map shows the propensity of water and DMSO to coalesce when impacting a dry surface or a wet well. Drops with volumes under 1 μL and velocities under 1 m/s will coalesce with a surface or liquid pool. Faster-moving drops (2 m/s) coalesce at drop volumes below 50 nL.
No splashing occurs for drops and well fluids composed of typical assay reagents or solvents like DMSO for Wec below 50, as the drop will coalesce into the well fluid. 11,12 Drops of these fluids do not splash on dry surfaces until the Wec exceeds 80. 13,14 Figure 3 shows that low-nanoliter droplets of either DMSO or water (as well as any binary mixture of the two) are far from the regime where splashing would occur in either wet or dry acoustic transfers. For example, a 50-nL drop of DMSO traveling at 1 m/s when it meets the receiver well has a critical impact Weber number of 10. A 50-nL drop of water at 1 m/s has Wec = 6. The Wec grows as the square of the drop velocity but only the cube root of volume. Hence, low drop velocities and, to a lesser extent, the low drop volumes enabled by ADE prevent splashing when drops land in the receiving plate. The splash-free transfer eliminates a potential source of cross contamination.
Turning Plates Upside Down
Inverting a microplate is not a feature common to microplate handling equipment. The acoustic transfer instrument incorporates plate inversion to ease integration with plate handling systems. Figure 4 shows as an example the plate inversion automation method for the instrument from Picoliter that provides a nest for robotic loading of both source plate and receiver plate in upright configurations. The source plate is pulled directly into the machine. The receiver plate is inverted by rotating it 180 degrees before bringing the receiver plate inside the instrument for the transfer process. When the fluid transfer operation is completed, the receiver plate exits the instrument in an inverted configuration, is rotated to an upright orientation, and made available for transport by conventional equipment.
Automation of direct plate-to-plate transfer inverts receiver plate before and after transfer.
Will the liquid stay in the wells when being flipped over or when being shuttled around during the transfer process? A number of 384-well and 1536-well flat bottom microplates from Greiner-Bio-One (Lake Mary, FL) and Nalge-NUNC (Naperville, IL) were tested for common fluids expected in “wet” transfers in a precommercial compound reformatter (Picoliter, Sunnyvale, CA). Common assay solvent systems were transferred into well plates and then sealed with Biomek Aluminum Foil Lids (Beckman Instruments, Fullerton, CA). The sealed plates were each run through 10 transfer motion sequences as shown in Figure 4 where the droplet transfer was simulated from a source 384-well plate into either a 384-well receiver plate or 384 interleaved wells of a 1536-well plate. Plates are flipped along the short axis as shown in Figure 4 in approximately 2 s with accelerations under 0.01g. The receiving plate was moved in a start/stop manner with respect to the source plate. Realignment of each receiver well to its source well was accomplished in under 200 ms.
The seal is able to show clear evidence of wetting when the inverted solution comes out of the well plate. After the 10 motion cycles (about 5 min of x-y-z motion and inversions), the foil seal was carefully pealed and examined. No spillage was detected on the seal. A summary of plate types, well volumes, and capacities of the wells tested is shown in Table 1.
Volumes of common assay fluids held by surface tension in a well of an inverted microplate during automated plate inversion and plate-to-plate liquid transfer operations
PP = Greiner 384 polypropylene plate, #781 201.
PS = Nunc 384 polystyrene cat# 164 688 and for the 1536 microplate, Greiner 1536 High Base, cat# 78095.
COC = Greiner 384 cyclic olefin plate (COC) cat# 781 801.
Non-Invasive
It should be noted that this adaptation of acoustic ejection to the microplate has the additional advantage of being noninvasive, meaning there is no contact between the acoustic device and the solution being transferred. Hence, it eliminates a common cause of cross-contamination among samples caused by the transfer device and a need for disposables or washing steps, leading to savings in time and/or cost. Non-invasive transfer simplifies automation of materials flow to the liquid-handling instrument, as there are no consumables such as tips and no wash fluid. Also, as discussed above, the ability of acoustic drop transfers to have small volumes (low nanoliters), with relatively low droplet velocity (under 2 m/s) upon deposition in a well, results in an impact Weber number below the point of splashing, reducing another potential source of cross-contamination.
Compound Library Reformatting
The vast majority of the compounds are stored dissolved in DMSO as the solvent. Since DMSO is very hygroscopic, it will absorb water from the atmosphere, diluting the dissolved compounds as well as altering the physical properties of the liquid in the microplate well. Environmental conditions and well geometry impact the rate of water absorption and equilibrium. When exposed to 30% relative humidity, DMSO gains about 25% of its original weight in water.
Initial liquid-handling instruments based on Picoliter® acoustic droplet ejection technology operate in the volume range of 5 to 50 nL for compound reformatting from 384-and 1536-well SBS-standard flat-bottom plates and are able to adapt to various DMSO/water ratios automatically over the typical mixture range of pure DMSO to 70/30 DMSO/water. Such a device enables HTS laboratories to realize the cost savings that result from reducing assay volumes without sacrificing the quality of results.
Of particular concern in assay design is ensuring that the amount of the DMSO transferred with the library compound will not impact the assay result. Many cell assays are sensitive and will respond differently when exposed to DMSO concentration of over 0.1%. Hence, reduction of compound transfer volumes to 50 nL facilitates assays in 50 μL, suitable for low-volume 384-well microplates. Transfer volumes of 5 nL enable assays in 5 μL and fit within typical 1536-well capacities. By eliminating contact between the device and the sample as well as easily addressing a variety of microplate form factors, the compound reformatter reliably enables the reduction of assay volumes to 384- and 1536-well formats.
Acoustic ejection technology is compatible with other flat-bottomed storage containers, including removable tubes (see Fig. 5). The use of removable tubes for compound delivery has been a growing trend. To integrate with existing automation, these tubes are designed to be carried in frames that have exterior dimensions that conform to SBS microplate standards.
Removable tubes with flat bottoms are suitable for acoustic ejection. Here, a 50-pL droplet of DMSO emerges from an overfilled polypropylene tube (MSP 101–1; Matrical, Spokane, WA) with a 1.5-mm inner diameter designed for a 384-tube rack system. These racks can be moved by conventional automation hardware.
Range of Solvents
ADE is well suited to the handling of viscous as well as volatile and corrosive liquids. Volatility can be an issue for some conventional technologies, and, in particular, evaporation can deplete the volume of the next liquid transfer or result in precipitation of solutes around the transfer outlet. ADE does not have any nozzle so evaporation and/or precipitation do not cause any significant change in the behavior at the acoustic focal spot. Since the transducer and focusing element are isolated from the liquid to be transferred, only the container holding the liquid needs to be chemically compatible. This enables researchers to use more appropriate and novel chemistries for specific assays.
Surface tension and viscosity for solvents common to life sciences have viscosities between 0.3 and 10 centipoise and a wide range of surface tensions. Volatile solvents like acetone, acetonitrile, and ethanol, with low viscosity and surface tension are ejectable with acoustics.
Precision of Volume Transfer
Variability in Picoliter® acoustic dispensing has been tested for volume transfers from 5 to 50 nL of DMSO hydrated up to 30% and containing 0.15-mM flourescein. A drop from each well of a 384-well source microplate was transferred to a well of a receiver microplate. The receiver microplate wells were then filled with 100 μL of 10 mM NaOH using a Beckman FX (Beckman, Fullerton, CA) liquid-handling robot. The plates were then read with a SPECTRAFluor Plus from Tecan (Maennedorf, Switzerland) to determine volume based on the fluorescence count and constructed standards curves. The precision of this measurement process is about 1.5% CV.
Tests indicate the CV of acoustic transfers grows with increasing variability or uncertainty in the nature of the materials being ejected. Drop-to-drop repeatability from the same well typically has a CV below 2%. When moving from well to well in a microplate, the CV is higher in large part due to uncertainty in the acoustic properties of the fluid being ejected, and for HTS, the composition in question is water content of the DMSO. The “unknown” amount of DMSO leads to errors in the speed of sound in the well fluid and hence to errors in focusing the acoustic energy and in delivering power to the fluid surface.
Monitoring acoustic energy reflected by the well fluid can be used to assess water content in DMSO, and this will be discussed in more detail in a future publication. Acoustic determination of the DMSO hydration provides an upper bound on the transfer precision of 8% CV when the composition of the DMSO is otherwise unknown. Improvements in assessment methods, and/or the application of external knowledge of DMSO composition, reduce CVs to 4%. The ADE process CVs are summarized in Table 2.
Volume transfer precision for acoustic drop ejection is between 2% and 8% CV. Precision of transfer correlates with precision in the determination of well fluid composition (and hence its acoustic properties). The measurement process used for this study has a CV of 1.5%.
Conclusion
Acoustic droplet ejection provides a clean method for transfer of liquids from microplate to microplate. The method enables the low-nanoliter DMSO compound additions required to take the next step in miniaturizing assays used with higher density microplates or for a microliter-scale, cell-based assay requiring low DMSO concentrations.
Acoustic microplate transfers are automation-friendly, as the plate inversion capability is built into the system. Liquid transfer operations into inverted plates, either empty or filled, can be achieved due to the relative strength of surface tension forces at the small spatial scales of microplate wells and low-nanoliter droplets. Neither gravity nor inertial forces from droplets dislodge reagents or cause splashing. The direct transfer of droplets from source reservoirs (both wells and tubes) containing a variety of liquids to receiver wells without any invasive contact reduces the risk of contamination, the need for consumables, or any wash steps.
ADE has been shown to transfer fluids common to life science liquid handling. The level of variability in the fluid and the well plates impacts the coefficient of variation measured in well plate transfers. More uniform microplates and knowledge of the fluid in the wells drive CVs down to 2%. Higher variability in plates and fluid properties result in CVs that remain below 8%. These precision levels hold for 5-nL transfers and enable assays, including cell-based assays, to be performed in 1536-well microplate formats.
Acknowledgment
The above work on focused acoustic droplet ejection includes the contributions of many people at Picoliter. The authors would like to acknowledge their tremendous efforts in demonstrating and documenting the capability of focused acoustics in small volume liquid handling for the life sciences.