Multichannel automated syringe pipettors are ubiquitous in high-throughput molecular biology laboratories (1) and are capable of aspirating and dispensing sample volumes as low as 100 nL (www.artrobbinsinstruments.com), but encounter difficulties transferring such volumes to a dry microtiter plate or to a fluid sample without contact. At submicroliter scales surface tension dominates over gravity, and drops remain attached to the pipettor needles. Touch-off methods (contact of the needles with the target well or fluid after the dispense from the pipettor is complete, or direct contact between the needles and the plate during dispense) are typically used to transfer small volumes to dry plates, but such contact techniques are highly sensitive to tip cleanliness, alignment, length, and flexibility, and often result in high variability of the transferred volume. In cases where the target well is filled with fluid, contact methods raise concerns of cross-contamination between runs. Piezo electric, ink-jet, or solenoid-actuated devices capable of ejecting small volumes exist (2,3,4,; www.deerac.com ; www.cartesiantech.com ; www.cybio-ag.com ; www.gilson.com ; www.biodot.com ; www.auroradiscovery.com), but are expensive, often less flexible, and are typically limited to relatively few channels since each channel needs an individual actuator, while syringe pipettors are available up to 384 channels (www.artrobbinsinstruments.com).

We present a low cost and highly adaptable method of sample transfer for generic pipettors based on electrostatic forces. Metering of the sample is performed by the pipettor; however, an electric potential is applied between the pipettor needles and the target plate providing an electrostatic force that pulls the drops into the target well. We present results with common reagents dispensed using a Hydra® (Art Robbins Instruments, Sunnyvale, CA, USA) that show this to be effective for volumes as low as 100 nL.

The electrostatic device is composed of an adjustable high voltage power supply and a microtiter plate charger ((Figure 1)). The charger consists of an aluminum plate (the anode) partly encased in an acrylic shell. The target plate is placed onto the exposed face of the aluminum plate. As a safety precaution, the aluminum plate can be covered with a thin insulating coating to prevent user contact with the anode, without compromising the performance of the device. A full description of the device, including construction information and cost, is available in the supplementary material available online at www.BioTechniques.com .

Figure 1.

The Hydra-96 Microdispenser (Art Robbins Instruments), which was used for all experiments, is composed of 96 actuated pipettors arrayed on a standard microtiter plate spacing and, for these experiments, is fitted with two types of pipettor needles: (i) 48 Teflon®-coated stainless steel tips and (ii) 48 Teflon-coated Nitinol® tips (a nickel-titanium alloy). It should be noted that the methods and device we present can be used with any pipettor, consisting of any number of channels, provided that the tips are electrically conductive or that there is a ground path to the fluid inside the tip. For pipettors with disposable tips, use of carbon-filled tips is sufficient to meet this criterion.

Fluorescent dye was used to assess dispensing performance. Test solutions included: distilled water, 95% ethanol, 25% polyethylene glycol (PEG), and BigDye® Mix (Applied Biosystems, Foster City, CA, USA) (2:1:7 of BigDye Ready Reaction Premix, BigDye sequencing buffer, and distilled water respectively). Each solution was mixed with fluorescein at a concentration of 10 mg/mL. Multiple experiments were conducted with each solution at 100, 300, and 600 nL nominal volumes. Skirted 96-well, V-bottom, polypropylene microtiter plates (MJ Research Microseal® 96; Bio-Rad Laboratories, Hercules, CA, USA) were used for all experiments. After the sample was dispensed into the microtiter plate, the plate was centrifuged for 1 min at 2755× g prior to being imaged on an AlphaImager® 3400 (Alpha Innotech Corporation, San Leandro, CA, USA). Experiments were performed confirming that the pH of the fluorescein solution (which impacts its quantum yield) remained stable, within 0.5 pH units, when exposed to air for the duration of the experiment.

Both wet-well and dry-well dispensing tests were performed. In wet-well tests, the target wells were prefilled with 10 µL of the same solution as was being dispensed (without fluorescein). In the case of BigDye Mix, the 10-µL target solution was distilled water. This volume was chosen because it is representative of volumes used in high-throughput molecular biology. However, greater or smaller volumes would not affect the performance of the electrostatic device, as the applied potential can be adjusted to compensate for a varying air gap between the fluid and the pipet tip. It should be noted that the target fluid volume is a low-impedance part of the electric circuit, and only the size of the air gap above the volume, rather than the height of the fluid column, affects the electric potential required for drop transfer.