Introduction

Growing needs to fill the drug discovery pipelines with new candidates have prompted changes in the screening process to increase speed and efficiency, while reducing costs. Emerging technologies in assay miniaturization, for high-throughput and ultra high-throughput screening of thousands of compounds, have become an important part of the ever changing drug discovery landscape. With limited material amounts as well as, increasing costs for development and storage of these compound libraries, liquid handling technologies become vital to reduce library usage, while ensuring the highest assay performance, throughput, and data quality.

Moreover, screening campaigns require the ability to support a wide range of sample/reagent volumes, from nanoliters to microliters, using the same automation platform. To address these issues, PerkinElmer has developed the P1 384 MDT NanoHead, with Modular Dispense Technology® (MDT) – a flexible pipetting solution for uHTS applications. NanoHead provides an optimal solution for plate replication, assay minia-turization, and protocols using challenging sample types such as DMSO as well as, protein and nucleic acid-containing solutions. The MDT NanoHead, in conjunction with three other MDT heads, permits automated pipetting from 96- to 1536-well plates, across a volume range of 50 nL to 235 µL.

This application note presents performance capabilities of PerkinElmer’s P1 384 MDT NanoHead pipetting tool for PerkinElmer automated liquid handling workstations. NanoHead performance data includes wet and dry pipetting precision, pipetting accuracy, plate reformatting, sample carryover, and demonstration of pipetting performance as used in a typical buffer/compound application. The following three case studies illustrate the features and advantages of PerkinElmer's MDT NanoHead.

Application Workstation

a P P l i c a T i o N N o T e

Authors

Nicole Deshong2,

Lee Brady1,

Jennifer Gale3,

David Bourdon, Ph.D., and

Gary Reznik, Ph.D.

1 PerkinElmer, Inc. Downers Grove, IL 60515 USA

2 PerkinElmer, Inc. Shelton, CT 06484 USA

3 Wyeth Research Collegeville, PA 19426 USA

NanoHead with Modular Dispense Technology for Flexible and Precise High-throughput, Fixed-tip Nanoliter Pipetting

2

Case Study 1: NanoHead 384-well wet versus dry dispensing

Materials and Methods: NanoHead wet and dry dis-pensing performance was evaluated using a PerkinElmer automated liquid handling workstation. Orange-G dye solution (Sigma, 2% w/v in DMSO) was dispensed at volumes ranging from 50 – 1000 nL. Destination plates (PerkinElmer 384-well black OptiPlates™) were filled with water prior to dispense (wet dispense condition) or post dye dispense (dry dispense), up to a final volume of 1 uL using the PerkinElmer FlexDrop™ Plus Precision Reagent Dispenser. Dry dispenses were fol-lowed by a 4-side tip touch. Absorbance readings were obtained using a PerkinElmer EnVision® Multilabel Plate Reader with resulting data reported as Dispense CV (%) across 384-channels.

Results and Discussion: The ability to “dry dispense” compounds prior to the addition of media or reaction buffer allows users to prepare plates in advance and store them until needed. Evaluations of Orange-G dye transfers demonstrate similarly high precision for both wet and dry dispenses across the indicated volume range (Figure 1).

Case Study 2: NanoHead sample carryover measurement

Materials and Methods: Sample carryover was measured between two liquid transfers in OptiPlate-384 black plates, with a tip wash in between. Blank fluo-rescence in OptiPlates was measured in an EnVision® Multilabel Plate Reader prior to dispensing. Two 384-well source plates were prepared: one containing 80 uL/well 1 mM fluorescein isothiocyanate (FITC, 80% DMSO, Source Plate 1), and the other containing 80 uL/well 1X phosphate buffered saline (PBS, Source Plate 2). Two 384-well destination plates were pre-pared, each containing 80 uL/well 1X PBS (Destination Plate 1 and Destination Plate 2). Using the NanoHead with hydrophobic tip coating, 500 nL of 1 mM FITC from Source Plate 1 was transferred (blowout mode) to Destination Plate 1. The NanoHead was then washed in the ultrasonic washbowl for approximately 60 seconds (5 aspirate-dispense cycles) using a 50% ethanol-50% water wash solution. Following the ultrasonic tip wash, 80 uL of 1X PBS was transferred from Source Plate 2 into Destination Plate 2. Residual FITC not removed during the wash cycle (and thus transferred into Destination Plate 2) was detected in the EnVision. In a separate procedure, a FITC concentration calibration curve was generated. Following background subtrac-tion, the calibration curve was used to interpolate

FITC concentrations into the wells of Destination Plate 2. The expected carryover concentration limit was 0.01% of the 1 mM FITC in Source Plate 1.

Results and Discussion: Proprietary tip coatings on the NanoHead provide significant advances in ultra-low volume dispensing performance with accuracy and precision for a number of sample types. FITC/DMSO carryover studies indicate that near background detectable levels of FITC fluorescence were observed as carryover following a sample transfer (Figure 2).

Figure 1. Comparison of NanoHead wet and dry pipetting precision over a range of volumes. Data represents dispense CV’s for wet or dry (with tip touch) 384-well dispensing of Orange-G dye (80% DMSO) using a PerkinElmer automated liquid handling workstation.

Figure 2. Sample carryover in the NanoHead following wash cycles with the ultrasonic washbowl. Concentrations of FITC/DMSO sample carryover were determined with the FITC fluorescence calibration curve (inset) and reported as a percentage of the original 1 mM source compound. The expected carryover limit of 0.01% is indicated in red.

3

Case Study 3: NanoHead and P30 384 MDT head compound transfer application

Materials and Methods: Pipetting precision for a typical compound transfer and dilution was evaluated using the P30 384 MDT and NanoHead pipetting heads docked on a single PerkinElmer automated liquid handling workstation deck. Two buffer transfers (7.5 uL and 15 uL) simulating compound diluent were performed using the P30 head and 100 nM rhodamine in 50 mM HEPES, pH 7.5. Compound dispenses (150 nL 10 µM rhodamine, 100% DMSO) were evaluated using the NanoHead. For NanoHead pipetting tests, destination plates were prepared with 15 uL of 50 mM HEPES buffer using the P30 MDT pipetting head. All plate evaluations were performed in triplicate for each volume/dispense head tested. Fluorescence readings were obtained on an EnVision, with resulting data reported as Dispense CV (%) across 384 channels. Data was provided courtesy of Wyeth Research.

Results and Discussion: Maintenance and testing of drug candidate libraries involves buffer/compound transfer series spanning a large volume range. Demonstrating the utility and flexibility of a single PerkinElmer automated liquid handling platform equipped with interchangeable docked NanoHead and P30 MDT heads, dual liquid transfers were performed with head swaps. Low CV values of less than 6% and 4% were observed on the NanoHead and P30 MDT head, respectively (Figure 3).

Conclusions

• TheP1 384 MDT NanoHead provides reliable pipetting accuracy as well as, precision at nanoliter volumes; compatible with all MDT-equipped PerkinElmer automated liquid handling workstations.

• Proprietarytipcoatingsandcleaningwiththeultrasonic washbowl minimizes sample carryover for reliable and cost-effective low volume liquid dispensing.

• NanoHead’sfunctionalpipettingrange(50nL–1000 nL) allows users to miniaturize assays and scale them as needed. Assay miniaturization can result in time savings as well as, reduced costs associated with reagents, consumables, and labor, while preserving valuable compounds.

• Whetherpreparinghigh-densitycompoundplates,plate reformatting (384- to 1536-well), or performing studies requiring dry dispense capabilities, NanoHead flexibility meets the needs of a variety of uHTS applications.

• AllMDTpipettingheadsfeature“activeidentification”providing automated, “on-the-fly” and error-free head swaps with individualized head recognition (i.e. storing individual calibrations and performance files). Fully automated and free of assay interruption, Modular Dispense Technology allows users to go from nanoliters to milliliters and/from 96- to 1536-well plates in less than one minute.

References

1. Gale, J. (2008) Target Class Specific Compound Selectivity Profiling. Podium Presentation: LabAutomation Annual Meeting – Palm Springs, CA

Figure 3. Dispense CV’s for a PerkinElmer automated liquid handling workstation with interchangeable NanoHead and P30 MDT dispense heads. Data represents CV values across 384-wells for compound/buffer transfer series using HEPES buffer (P30 MDT dispenses at 7.5 or 15 uL using P30 tips) and compound transfers using DMSO (NanoHead dispenses at 150 nL). Data are mean ± S.E.M. of three independent experiments for each volume/head type. Data courtesy of Wyeth Research [1].

For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs

Copyright ©2009, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 008284A_01 Printed in USA

PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602www.perkinelmer.com