Abstract
INTRODUCTION
Assay development is often facilitated by testing the chemistry against a representative or focused library of molecular compounds. Boston Innovation Inc. (“BII”), has introduced its SmartPlate technology which integrates molecular compound storage and dispensing in a single tool which enables many assay iterations quickly, easily, and at low cost, while maintaining consistent compound integrity. SmartPlate does this by storing concentrated compounds in 100% DMSO in sealed reser-voirs, each with a dedicated dispensing tap. The compounds are never exposed to air, moisture, or light. Risks of cross-contamination and the need for cleaning or disposing pipette tips are elimi-nated. Compounds are metered and integrally diluted within the dispensing taps using BII's Direct Dilution™ technology, which allows SmartPlate to serve the needs of high density assay platforms with non-contact dispensing from 5 to 200nL of compound directly to standard assay plates. The integration of functions cuts waste, cost, and time from the drug discovery process by maintaining com-pound integrity, preserving valuable compound inventory, and elim-inating process steps. At 15nL compound dispensing, 15μL of each compound in SmartPlate can now theoretically support one thou-sand assays without revisiting the main compound repository.
DEVELOPMENT TIMELINE
In 1999, Boston Innovation Inc. (BII), Cambridge, MA introduced a new technology for non-contact dispensing sub-10μL volumes of fluid. Encapsulated in that invention was the integration of a small reservoir and a dedicated dispensing tap. Commencing with the Screentech 1999 conference, BII began its focus on solving the critical problem of dispensing small amounts of compound into assays while preserving compound inventory.
Over the following few months, BII assembled a Scientific Advisory Board from a variety of pharmaceutical laboratories to define a specification for a plate-based device that combined sealed reservoirs with dedicated dispenser taps. The specification requirements included three main feature sets: shipping, storing, and dispensing of liquid compounds. Shipping or portability was desired, enabling decentralization of all or a portion of compound libraries and facilitating exchange between laboratories or between work stations. Storage was included so that compounds could be kept in this new plate format for extended periods of time, while preserving compound integrity. Finally, and perhaps the most challenging specification, was integrated dispensing for laboratory efficiency.
BII first focused its attention on developing a new technology capable of non-contact dispensing 100nL volumes of DMSO dissolved compound directly to assay sites, which was first demonstrated a year ago at the Drug Discovery Technology 2000 conference. Last summer, the SmartPlate prototype was introduced demonstrating 15nL non-contact dispensing. BII is soon introducing SmartPlate384β, a 384-channel product facilitating improved assay development.
SMARTPLATE SPECIFICATION AND TECHNICAL DESCRIPTION
Traditionally, compounds are stored in 96-well or 384-well polypropylene or polystyrene microtiter plates with a variety of covers or seals. These plates are stored at room temperature, 4°C or perhaps frozen to −20°C. The microtiter plates are removed from storage, opened, and DMSO dissolved compounds are aspirated via a pipettor. Studies have shown that compounds in source plates suffer degradation when they experience multiple freeze/thaw and open/reseal cycles. Exposure to air and moisture can cause compounds to “crash” out of solution or lose their potency. Molar concentration of a compound after a number of thermal cycles and environmental exposure is difficult to maintain and can vary significantly.
SmartPlate stores 3–20μl of DMSO dissolved compounds in sealed reservoirs that are backed up with an inert gas (Figure 1) minimizing any DMSO absorption of moisture. The sealed reservoirs are designed to accommodate temperature and pressure changes and protect the compound from light and contamination. The SmartPlate is constructed out of molded and extruded materials made from polypropylene and Teflon coated silicone. The dispensing tube shown in Figure 1 is a dedicated dispenser tap and all 96 or 384 dispenser taps are attached to a translating tube plate.
The operating sequence of SmartPlate begins with Figure 2.
SmartPlate mounts to a SmartManifold which is attached to and existing pipettor. Assay plates pass underneath to be filled.
Traditional plate handling automation delivers a SmartPlate to the SmartPlate module which is comprised of an existing pipettor with a proprietary manifold (“SmartManifold”) and a buffer reservoir. In Figure 3, the tube plate is translated up, opening a vent to a dry, inert gas source. A small capillary hole in the side of the tube translates through the lower seal and into the liquid compound reservoir. Capillary forces pull the compound into the capillary hole. This capillary metering method enables precise and accurate transfer of sub-100nL volumes out of the reservoir.
SmartPlate in capillary fill position.
In Figure 4, the tube plate has translated back down effectively metering the liquid compound from the sealed reservoir. The SmartManifold directs a jet of air (or N2) at the capillary hole emptying the fluid into the inner diameter of the tube.
Resealed SmartPlate with filled capillary. Compound in metering capillary is then blown to inside surface of dispensing tube.
In Figure 5, with the aid of the pipettor, the SmartManifold delivers 1–10μL of buffer to each channel. As the buffer bolus translating down the dispensing tube reaches the compound that was blown into the tube, the fluids mix. The buffer-compound mixture is then presented in full or in part to the tip of the tube. The SmartManifold then dispenses the mixture non-contact directly into any assay site. This process is deemed Direct Dilution™.
Direct Dilution. A bolus of buffer comes down from the manifold through the dispensing tube, mixes, and is then dispensed.
The SmartManifold provides additional system flexibility. For example, a laboratory has a set of compounds residing in SmartPlates with 15nL capillary holes. For a given assay, the requirements dictate a compound addition of 50nL. To achieve this, as shown in Figure 4, the tube translation capillary blow-in sequence is repeated four times resulting in 60nL of compound residing inside the tube. Following in Figure 5, the SmartManifold delivers 6μL of buffer to each dispense tube, mixes the buffer with the compound and then dispenses only 5μL, resulting in a dispensed mixture of 50nL of compound and 5μL of buffer into each assay site.
Before the SmartPlates are sent back to the library storage site, they pass through a rinse station. A mild solvent or aqueous solution rinses each tube to eliminate any traces of buffer or compound.
TEST RESULTS
Two tests were performed at a pharmaceutical laboratory with a SmartPlate eight-channel prototype device. All test dispensing was non-contact into small volume 384-well microtiter plates.
The first test was designed to determine precision and accuracy when dispensing DMSO with fluorescein dye. 15nL was non-contact dispensed simultaneously from eight individually sealed channels with 6μL of buffer into a dry plate. 94μL of buffer was added to each plate well and the signal measured with an LJL Analyst 96/384. Dispensed CV's for 15nL ranged from 1.6% to 3.6% with a full plate CV of 8.47%. There were no unexpected errors.
The second test was designed to determine the precision and accuracy of dispensing inhibitor into an assay with a total volume of 12μL. Assay plates were pre-filled with 3μL of enzyme and 3μL of substrate. 15nL was non-contact dispensed simultaneously from 8 individually sealed channels with 6μL of buffer into the assay plates.
One plate was incubated for 1.5 hours for an inhibition of 38.7% and the other plate was incubated for 2 hours for an inhibition of 45.5%. Inhibition CV's ranged from 4% to 8.9% by tip with full plate CV's of 10.4% and 8.5% respectively. Total signal and background CV's ranged from 6.3% to 7.7%
The conclusion from these tests is that SmartPlate technology is able to dispense low nanoliter volumes in either wet or dry plates with sub-10% CV's.
PROCESS FLEXIBILITY
SmartPlate technology is useful for many aspects of drug discovery. Representative, mirror, or pilot libraries can be packaged to support assay development (see first product offering below). Reservoir capacity enables compound storage, while reducing the need to revisit master plates, enabling current compound inventory to last longer. Resynthesizing compounds is required less often. The combined reservoir and Direct Dilution dispensing technology enables single step high throughput screening. And lastly, secondary screening is supported through SmartPlate cherry picking capability, as all SmartPlate reservoirs and taps are individually addressable. An SmartPlate system designed for cherry picking can pull 3000 picks in five hours.
The features described in this article provide, among others, the following benefits:
Traditional HTS dilution and reformatting steps are eliminated.
Cross tip and cross well contamination are eliminated.
Compound can be delivered to assays requiring as little as 5nL or as much as 200nL.
Sub 20μL assays are supported.
Dispense dead volume is zero.
SmartPlate delivers unparalleled library utilization and preserves central store.
Compounds can be transported, stored, and dispensed with ease for process flexibility.
SYSTEM IMPLEMENTATION
SmartPlate technology is most useful when configured as a system connecting a compound library storage chamber through a server to a High Throughput Screening (“HTS”) assay work station. The following schematic, Figure 6, is a basic integrated system utilizing a plate storage chamber, plate moving robotics, a buffer reservoir, a HTS work station, and a SmartPlate rinse station. SmartPlates will cycle out of storage and run parallel with assay plates, dispense compound, move to a rinse station, and return to storage.
SmartPlate system implementation.
Rendering of 384 well Smartplate.
SMARTPLATE384β SYSTEM PRODUCT INTRODUCTION
Boston Innovation is introducing its first product, SmartPlate384β. The system, aimed at small libraries, will provide enough SmartPlates to package 5,000 – 15,000 compounds and will include all the functional modules, making the SmartPlate384β a stand-alone workstation. This workstation is suitable for representative, diverse, or pilot libraries that are accessed many times a month for assay development. The SmartPlate384β will provide an assay ready copy of 10,000 compounds in approximately 15 minutes. In future systems, SmartPlate compound inventory will be tracked with standard bar code or dot code technology.
For more information, please contact Jeffrey A. Karg at
Jeffrey A. Karg
